The majority of BRCA1-associated breast cancers are basal cell-like, which is associated with a poor outcome. Using a spontaneous mouse mammary tumor model, we show that platinum compounds, which generate DNA breaks during the repair process, are more effective than doxorubicin in Brca1/p53-mutated tumors. At 0.5 mg/kg of daily cisplatin treatment, 80% primary tumors (n = 8) show complete pathologic response. At greater dosages, 100% show complete response (n = 19). However, after 2 to 3 months of complete remission following platinum treatment, tumors relapse and become refractory to successive rounds of treatment. Approximately 3.8% to 8.0% (mean, 5.9%) of tumor cells express the normal mammary stem cell markers, CD29 hi 24 med , and these cells are tumorigenic, whereas CD29 med 24 À/lo and CD29 med 24 hi cells have diminished tumorigenicity or are nontumorigenic, respectively. In partially platinum-responsive primary transplants, 6.6% to 11.0% (mean, 8.8%) tumor cells are CD29 hi 24 med ; these populations significantly increase to 16.5% to 29.2% (mean, 22.8%; P < 0.05) in platinum-refractory secondary tumor transplants. Further, refractory tumor cells have greater colony-forming ability than the primary transplant-derived cells in the presence of cisplatin. Expression of a normal stem cell marker, Nanog, is decreased in the CD29 hi 24 med populations in the secondary transplants. Top2A expression is also down-regulated in secondary drug-resistant tumor populations and, in one case, was accompanied by genomic deletion of Top2A . These studies identify distinct cancer cell populations for therapeutic targeting in breast cancer and implicate clonal evolution and expansion of cancer stem-like cells as a potential cause of chemoresistance. [Cancer Res 2008;68(9):3243-50]

The continued threat of SARS-CoV-2 to global health necessitates development of improved research tools and vaccines. We present an improved SARS-CoV-2 spike ectodomain, 'VFLIP', bearing five proline substitutions, a flexible cleavage site linker, and an inter-protomer disulfide bond. VFLIP displays significantly improved stability, high-yield production and retains its trimeric state without exogenous trimerization motifs. High-resolution cryo-EM and glycan profiling reveal that the VFLIP quaternary structure and glycosylation mimic the native spike on the viral surface. Further, VFLIP has enhanced affinity and binding kinetics relative to other stabilized spike proteins for antibodies in the Coronavirus Immunotherapeutic Consortium (CoVIC), and mice immunized with VFLIP exhibit potent neutralizing antibody responses against wild-type and B.1.351 live SARS-CoV-2. Taken together, VFLIP represents an improved tool for diagnostics, structural biology, antibody discovery, and vaccine design.

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

Apoptosis was detected in Vero cell cultures expressing transfected dengue virus type 2 (DENV-2) genes. Approximately 17?5 and 51?5 % of cells expressing NS3 serine protease and NS2B-NS3 185 serine protease precursor protein [NS2B-NS3 185 (pro)] genes, respectively, were apoptotic. The percentage of apoptotic cells was significantly higher in cell cultures expressing NS2B-NS3 185 (pro). NS2B-NS3 185 (pro) was detected as NS2B-NS3 185 (pro)-EGFP fusion protein in cytoplasmic vesicular structures in the apoptotic cells. Site-directed mutagenesis which replaced His 51 with Ala within the protease catalytic triad significantly reduced the ability of the expressed NS3 and NS2B-NS3 185 (pro) to induce apoptosis. Results from the present study showed that DENV-2-encoded NS3 serine protease induces apoptosis, which is enhanced in cells expressing its precursor, NS2B-NS3 185 (pro). These findings suggest the importance of NS2B as a cofactor to NS3 protease-induced apoptosis.Dengue is a mosquito-borne disease endemic in many Southeast Asian countries. The disease is caused by Dengue virus (DENV), a positive single-stranded RNA virus belonging to the Flaviviridae family. There are four antigenically related but distinct DENV serotypes; DENV-1 to -4, all of which can cause dengue fever and the more severe forms of the disease, dengue haemorrhagic fever and dengue shock syndrome (Henchal & Putnak, 1990). The mechanism leading to the severe manifestation of dengue is still unknown. In recent years, however, a number of studies have shown that infection with DENV induces apoptosis in vitro and also in vivo (Despres et al., 1996;Marianneau et al., 1997;Shafee & AbuBakar, 1997;Couvelard et al., 1999;Lei et al., 2001;Huerre et al., 2001). These studies raised the possibility that apoptosis is important in the pathogenesis of dengue. The mechanisms whereby DENV triggers the apoptotic cellular responses, however, have not been thoroughly investigated, though results from a recent study demonstrated the requirement for live virus for induction of apoptosis to occur (Shafee & AbuBakar, 2002). In various other systems, the involvement of serine proteases in the induction of apoptosis has been reported (Verhagen et al., 2002;Martins et al., 2002;Hegde et al., 2002). Serine proteases such as Omi/HtrA2 and Smac/ DIABLO have been shown to antagonize the inhibitors of apoptosis proteins (IAP), and thus to promote apoptosis. Among RNA viruses, it was shown that the NS3 protease of Langat virus (Prikhod'ko et al., 2002), and the 3C protease of Enterovirus 71 (Li et al., 2002) and Poliovirus (Barco et al., 2000), induced apoptosis in infected cells. In DENV infection, however, the potential involvement of DENV-2-encoded serine protease in the induction of apoptosis has not been investigated. In the present study, the effects of DENV-2 NS3 protease and NS2B-NS3 185 (pro) overexpression in transiently transfected Vero cells were investigated and compared with results obtained with other dengue virus genes.All DENV genes used in the present stu...