CIRP Expression on Growth and Productivity of CHO Cells

Mammalian cell culture is typically operated at the physiological temperature of 37°C. Low temperature cell culture at 30-33°C, in particular for CHO cells, increased the specific productivity of many recombinant proteins amongst many other benefits. However, the cell density is lower, thus reducing the total protein yield. Of the 17 mammalian cold-stress genes reported to be up- or down-regulated at low temperature, CIRP shows potential as a gene target for improving recombinant protein production, as its expression levels were reported to affect both growth and specific productivity. In this study, it was shown that over-expression of the cold-stress gene CIRP did not cause growth arrest in CHO cells, in contrast to previous reports. However, over-expression of CIRP successfully improved the specific productivity and total yield of a recombinant interferon-γ CHO cell-line at 37°C by 25%.Singapore-MIT Alliance (SMA

Enforcing Socioeconomic Rights: Everyday Agency, Resistance and Community Resources Among Indonesian Domestic Workers in Hong Kong

There are currently over 320,000 full-time, live-in migrant domestic workers (MDWs) in Hong Kong, the vast majority of whom are from Southeast Asia. In recent years, women from the Philippines and Indonesia have constituted 48 per cent and 49 per cent, respectively, of the MDW population, with women from Thailand, Sri Lanka and Nepal making up most of the remainder (Immigration Department 2012). These women and previous generations of women from Southeast Asia account for a significant proportion of the intra-Asia migration of workers, being at the same time a part of the widely observed feminization of migration (see also Elias and Louth, this volume). In the social fabric of Hong Kong, their arrival in ever-increasing numbers, especially since the second half of the 1980s, also marked a sea change in which household chores, child and elder care were commoditized to the extent that in approximately one in eight Hong Kong households an MDW carries out or helps with these tasks. The nature of domestic work, its private location and the dispersal of its workers, very often as single employees in a household, coalesce to produce a vulnerability that is, in many parts of Asia, worsened by live-in arrangements which may have an impact on access to unions or to help and advice. This vulnerability is why the International Labour Organization has identified women domestic workers as one of the three most vulnerable groups of migrant workers (ILO 2004) and why a Domestic Workers’ Convention has now been adopted (ILO number 189). This convention and the longer-standing UN Convention on the Protection of the Rights of All Migrant Workers and Members of their Families (1990) have had some indirect impact in Hong Kong in terms of consciousness raising and lobbying. NGOs and migrant workers’ unions, which in Hong Kong are permitted the political space in which to establish themselves and pursue their activities, have invoked both conventions in their ongoing campaigns and, arguably, drawn legitimacy from them in fighting two recent major issues: the exclusion of MDWs from the right of abode (akin to permanent residency) (Vallejos and Domingo v Commissioner of Registration 2011) and the exclusion of domestic work from the minimum wage regulations

Cell-line Engineering of Chinese Hamster Ovary Cells for Low-temperature Culture

Developments in mammalian cell culture and recombinant technology has allowed for the production of recombinant proteins for use as human therapeutics. Mammalian cell culture is typically operated at the physiological temperature of 37°. However, recent research has shown that the use of low-temperature conditions (30-33°) as a platform for cell-culture results in changes in cell characteristics, such as increased specific productivity and extended periods of cell viability, that can potentially improve the production of recombinant proteins. Furthermore, many recent reports have focused on investigating low-temperature mammalian cell culture of Chinese hamster ovary (CHO) cells, one of the principal cell-lines used in industrial production of recombinant proteins. Exposure to low ambient temperatures exerts an external stress on all living cells, and elicits a cellular response. This cold-stress response has been observed in bacteria, plants and mammals, and is regulated at the gene level. The exact genes and molecular mechanisms involved in the cold-stress response in prokaryotes and plants have been well studied. There are also various reports that detail the modification of cold-stress genes to improve the characteristics of bacteria or plant cells at low temperatures. However, there is very limited information on mammalian cold-stress genes or the related pathways governing the mammalian cold-stress response. This project seeks to investigate and characterise cold-stress genes that are differentially expressed during low-temperature culture of CHO cells, and to relate them to the various changes in cell characteristics observed in low-temperature culture of CHO cells. The gene information can then be used to modify CHO cell-lines for improved performance in the production of recombinant proteins.Singapore-MIT Alliance (SMA

High Performance Substrate Integrated Waveguide Bandstop Filter using Dual-radial Cavity Resonator

A SIW bandstop filter is designed by coupling two cavity resonators to the SIW line. Dual-radial cavity resonators are proposed to design the SIW bandstop filter. Two single radial cavity resonator SIW bandstop filter are designed first. After that, a dual-radial cavity resonator SIW bandstop filter is designed based on the two single radial cavity resonator SIW bandstop filter. The designed bandstop filter operates at X-band frequency with a stopband from 8.7GHz to 9.2GHz, and it is implemented on RO4350B substrate with a thickness of 0.508mm. An insertion loss of less than 3dB is achieved, and the bandstop filter have a sharp roll of with attenuation of -34dB. This bandstop filter can be applied in the radar syste

Law and the administration of justice in the British leased territory of Weihaiwei.

The constitution of the British-leased territory of Weihaiwei (1898-1930) introduced a legal system which turned out to be too elaborate for the territory's needs; there was never a resident judge or barrister and most cases were heard and investigated by nonspecialist government administrators. With the exception of civil disputes between Chinese, the courts were to apply English law and procedure. Most accused and litigants, however, had their cases heard in accordance with laws and procedures which were quite different from those used in England. Defendants in criminal cases were tried by lay magistrates without a lawyer; rights in civil cases were determined by Chinese law; and headmen supplemented the police in maintaining order in the villages. When it was discovered that the appeals system had not been used, a simpler procedure was introduced. On the whole, the authorities prioritised the hearing of civil disputes, the involvement of headmen in law reform, mediation and law enforcement. In contrast, they were indifferent towards jury trials and lawyers, and reluctant to pursue reforms ahead of social change. Indeed, success in providing access to the courts for civil disputes inadvertently undermined traditional mediation. When it came to a social problem such as suicide, the authorities, though remarkably well informed about suicide amongst the Chinese, tackled only its aftermath. Although new, the legal system which affected the territory's Chinese inhabitants was not entirely unfamiliar; by their position and functions, the magistrates resembled the Chinese district magistrate; Chinese law was often applied; some civil and criminal cases were tried by headmen; and village regulations were recognised. It was a legal system shaped not only by the conservatism of individual officials but also by factors such as Weihaiwei's unpromising start, its subsequent decline in strategic importance, demography, shortage of officials, and lack of socio-economic development

The Benefits of Combined Anti-platelet Treatment in Carotid Artery Stenting

AbstractObjectiveTo assess the benefits of a combined anti-platelet regime of aspirin and clopidogrel in carotid artery stenting.MethodsA randomised controlled trial was performed comparing aspirin and 24-h heparin with aspirin and clopidogrel for patients undergoing carotid artery stenting. Outcome measures included 30-day bleeding and neurological complications and 30-day stenosis rates.ResultsBleeding complications (groin haematoma or excessive bleeding at the groin site) occurred in 17% of the heparin and 9% of the clopidogrel group (p=0.35; n.s). The neurological complication rate in the 24-h heparin group was 25% compared to 0% in the clopidogrel group (p=0.02). The 30-day 50–100% stenosis rates were 26% in the heparin group and 5% in the clopidogrel group (p=0.10; n.s).ConclusionsThe dual anti-platelet regime has a significant impact on reducing adverse neurological outcomes without an additional increase in bleeding complications. This study was terminated prematurely due to an unacceptable level of complications in the heparin arm of the trial

Enhancing Production of Recombinant Proteins from Mammalian Cells

The bio-manufacturing of recombinant proteins from mammalian cell cultures requires robust processes that can maximize protein yield while ensuring the efficacy of these proteins as human therapeutics. Recognizing that the challenge of improving protein yield and quality can be met through various approaches, this paper presents three strategies currently being developed in our group. A method for rapidly selecting subpopulations of cells with high production characteristics is proposed. This method combines the efficiency of green fluorescent protein/fluorescence-activated cell sorting (GFP/FACS)–based screening with homologous recombination to generate and select high-producing subclones. Next, the development of chemically defined, protein-free media for enhancing monoclonal antibody production is described. Analysis of culture media effects on the genome-wide transcriptional program of the cell is presented as a means to optimize the culture media and identify potential targets for genetic manipulation. Finally, we propose a method for increasing the extent of intracellular sialylation by improving the transport of CMP-sialic acid into the trans-Golgi. This is hypothesized to increase the sialic acid availability, and may enhance the degree of sialylation in the glycoprotein product.Singapore-MIT Alliance (SMA

Quantum interference in the fluorescence of a molecular system

It has been observed experimentally [H.R. Xia, C.Y. Ye, and S.Y. Zhu, Phys. Rev. Lett. {\bf 77}, 1032 (1996)] that quantum interference between two molecular transitions can lead to a suppression or enhancement of spontaneous emission. This is manifested in the fluorescent intensity as a function of the detuning of the driving field from the two-photon resonance condition. Here we present a theory which explains the observed variation of the number of peaks with the mutual polarization of the molecular transition dipole moments. Using master equation techniques we calculate analytically as well as numerically the steady-state fluorescence, and find that the number of peaks depends on the excitation process. If the molecule is driven to the upper levels by a two-photon process, the fluorescent intensity consists of two peaks regardless of the mutual polarization of the transition dipole moments. If the excitation process is composed of both a two-step one-photon process and a one-step, two-photon process, then there are two peaks on transitions with parallel dipole moments and three peaks on transitions with antiparallel dipole moments. This latter case is in excellent agreement with the experiment.Comment: 11 pages, including 8 figure

Hydrokinetic pancreatic function and insulin secretion are moduled by Cl− uniporter Slc26a9 in mice

Aim: Slc26a9 is a member of the Slc26 multifunctional anion transporter family. Polymorphisms in Slc26a9 are associated with an increased incidence of meconium ileus and diabetes in cystic fibrosis patients. We investigated the expression of Slc26a9 in the murine pancreatic ducts, islets and parenchyma, and elucidated its role in pancreatic ductal electrolyte and fluid secretion and endocrine function. Methods: Pancreatic Slc26a9 and CFTR mRNA expression, fluid and bicarbonate secretion were assessed in slc26a9−/− mice and their age- and sex-matched wild-type (wt) littermates. Glucose and insulin tolerance tests were performed. Results: Compared with stomach, the mRNA expression of Slc26a9 was low in pancreatic parenchyma, 20-fold higher in microdissected pancreatic ducts than parenchyma, and very low in islets. CFTR mRNA was ~10 fold higher than Slc26a9 mRNA expression in each pancreatic cell type. Significantly reduced pancreatic fluid secretory rates and impaired glucose tolerance were observed in female slc26a9−/− mice, whereas alterations in male mice did not reach statistical significance. No significant difference was observed in peripheral insulin resistance in slc26a9−/− compared to sex- and aged-matched wt controls. In contrast, isolated slc26a9−/− islets in short term culture displayed no difference in insulin content, but a significantly reduced glucose-stimulated insulin secretion compared to age- and sex-matched wt islets, suggesting that the impaired glucose tolerance in the absence of Slc26a9 expression these is a pancreatic defect. Conclusions: Deletion of Slc26a9 is associated with a reduction in pancreatic fluid secretion and impaired glucose tolerance in female mice. The results underline the importance of Slc26a9 in pancreatic physiology. © 2021 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society

Cell-line Engineering for Low-temperature Growth

In the chemical industry, the rates of reactions are usually enhanced by the use of high-temperature and high-pressure conditions. This chemical engineering approach is rarely applied in the biotechnology field. Firstly, most biochemical reactions take place in an aqueous phase, which makes them relatively insensitive to changes in pressure. Secondly, they form a tightly regulated network, with distinct pathways that operate optimally at relatively low temperatures of about of 25-40°C. Beyond this range, higher temperatures would denature the proteins in the cell, leading to eventual cell death. Mammalian cells are integral the biotechnology field for production of human therapeutics. Bio-reactors for mammalian cells are typically operated at 370C. The effects of temperature down-shifting have been well-investigated and documented for several mammalian cell-lines and recombinant products. Although the rate of growth of cells is reduced, the productivity of recombinant protein is increased at lower temperatures. Apoptosis and nutrient requirements are reportedly reduced at lower temperatures also. As such, it is advantageous to investigate the effects of mammalian cell culture at down-shifted temperatures, with the ultimate aim of improving recombinant protein production and quality.Singapore-MIT Alliance (SMA