Functional characterization of sensory rhodopsin II from Halobacterium salinarum expressed in Escherichia coli

Sensory rhodopsin II (SRII) from Halobacterium salinarum is heterologously expressed in Escherichia coli with a yield of 3-4 mg of purified SRII per liter cell culture. UV/Vis absorption spectroscopy display bands characteristic for native SRII. The resonance Raman spectrum provides evidence for a strongly hydrogen-bonded Schiff base like in mammalian rhodopsin but unlike to the homologous pSRII from Natronobacterium pharaonis. Laser flash spectroscopy indicates that SRII in detergent as well as after reconstitution into polar lipids shows its typical photochemical properties with prolonged photocycle kinetics. The first functional heterologous expression of SRII from H. salinarum provides the basis for studies with its cognate transducer HtrII to investigate the molecular processes involved in phototransduction as well as in chemotransduction.     

Design and characterization of viral polypeptide inhibitors targeting Newcastle disease virus fusion

Paramyxovirus infections can be detected worldwide with some emerging zoonotic viruses and currently there are no specific therapeutic treatments or vaccines available for many of these diseases. Recent studies have demonstrated that peptides derived from the two heptad repeat regions (HR1 and HR2) of paramyxovirus fusion proteins could be used as inhibitors of virus fusion. The mechanism underlying this activity is in accordance with that of class I virus fusion proteins, of which human immunodeficiency virus (HIV) and influenza virus fusion proteins are members. For class I virus fusion proteins, the HR1 fragment binds to HR2 to form a six-helix bundle with three HR1 fragments forming the central coiled bundle surrounded by three coiled HR2 fragments in the post fusion conformational state (fusion core). It is hypothesized that the introduced exogenous HR1 or HR2 can compete against their endogenous counterparts, which results in fusion inhibition. Using Newcastle disease virus (NDV) as a model, we designed several protein inhibitors, denoted HR212 as well asHR121 and 5-Helix, which could bind the HR1 or HR2 region of fusion protein, respectively. All the proteins were expressed and purified using a GST-fusion expression system in Escherichia coli. The HR212 or GST-HR212 protein, which binds the HR1 peptide in vitro, displayed inhibitory activity against NDV-mediated cell fusion, while the HR121 and 5-Helix proteins, which bind the HR2 peptide in vitro, inhibited virus fusion from the avirulent NDV strain when added before the cleavage of the fusion protein. These results showed that the designed HR212, HR121 or 5-Helix protein could serve as specific antiviral agents. These data provide additional insight into the difference between the virulent and avirulent strains of NDV.     

Effects of scavengers for active oxygen species on cell death by cryptogein

The hypersensitive reaction is a type of programmed cell death in plants. Cryptogein is a proteinaceous elicitor secreted from Phythophthora cryptogea. In one current model, active oxygen species (AOS) trigger programmed cell death in plants. In this study, we examined a variety of AOS scavengers to elucidate the function of AOS in the death program. Most of these AOS scavengers, including tiron, a scavenger for superoxide radical, catalase for hydrogen peroxide, and hydroquinone, sodium ascorbate and propyl gallate for free radicals, almost completely removed extracellular AOS. However, none of the reagents completely blocked the cell death process. Other reagents, such as histidine and dimethylfuran, scavengers for singlet oxygen, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, showed significant toxicity in BY-2 cells. These results indicate that AOS produced in the extracellular space do not play a role in hypersensitive cell death.     

Lobaplatin-based regimens outperform cisplatin for metastatic breast cancer after anthracyclines and taxanes treatment

The goal of this study was to assess the antitumor efficacy and safety of lobaplatin-based regimens as the second line of treatment in patients with metastatic breast cancer (MBC) resistant to anthracyclines and taxanes, compared with that of cisplatin-based regimens. During August 2012 to April 2015, 87 patients who received lobaplatin-based regimens or cisplatin-based regimens were included. Medical records of the patients noted that lobaplatin (30?mg/m²) or cisplatin (25?mg/m²), combined with another chemotherapeutic agent such as Gemcitabine (1000?mg/m²) or Vinorelbine (25?mg/m²), was intravenously given to the patients on a basis of twenty-one days as one treatment cycle. All the patients were followed until August 2017. The endpoint of this study was progression-free survival (PFS), overall survival (OS), and estimated objective response rate (RR). Safety and drug tolerability data were also obtained. Lobaplatin-based regimens prolonged PFS compared to cisplatin-based regimens (median 13.2 vs 4.7?months, hazard ratio?=?0.37, 95% confidence intervals: 0.21–0.67, P?=?.0007), while OS was not significantly different between the two groups (hazard ratio?=?0.72, 95% confidence intervals: 0.40–1.30, P?=?.2767), as was objective RR (37.8% vs 33.4%, $x^2$ = 0.19, P?=?.6653). Nausea/vomiting and renal injury were more frequent with cisplatin-based regimens. Our results show that lobaplatin-based regimens are superior to cisplatin in terms of efficacy and are better tolerated.     

Relationship between OPA1 and cardiolipin in mitochondrial inner-membrane fusion

Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. The large GTPase optic atrophy 1 (OPA1) is identified as a core component of inner membrane (IM) fusion. OPA1 exists as the membrane-anchored L-OPA1 and the proteolytically cleavage soluble S-OPA1. Recently, we showed that OPA1 and mitochondria-localized lipid cardiolipin (CL) cooperate in heterotypic IM fusion [Ban et al., Nat. Cell Biol. 19 (2017) 856–863]. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 and S-OPA1 expressed in silkworm and found that L-OPA1 on one side of the membrane and CL on the other side were sufficient for mitochondrial fusion. L-OPA1 is the major fusion-prone factor in heterotypic fusion. However, the role of S-OPA1 remains unknown as S-OPA1 promoted L-OPA1-dependent heterotypic membrane fusion and homotypic CL-containing membrane fusion, but S-OPA1 alone was not sufficient for heterotypic membrane fusion. L-OPA1- and CL-mediated heterotypic mitochondrial fusion was confirmed in living cells, but tafazzin (Taz1), the causal gene product of Barth syndrome, was not essential for mitochondrial fusion. Taz1-dependent CL maturation might have other roles in the remodeling of mitochondrial DNA nucleoids.     

BCL10 is recruited to sites of DNA damage to facilitate DNA double-strand break repair

Recent studies have found BCL10 can localize to the nucleus and that this is linked to tumor aggression and poorer prognosis. These studies suggest that BCL10 localization plays a novel role in the nucleus that may contribute to cellular transformation and carcinogenesis. In this study, we show that BCL10 functions as part of the DNA damage response (DDR). We found that BCL10 facilitates the rapid recruitment of RPA, BRCA1 and RAD51 to sites of DNA damage. Furthermore, we also found that ATM phosphorylates BCL10 in response to DNA damage. Functionally, BCL10 promoted DNA double-strand breaks repair, enhancing cell survival after DNA damage. Taken together our results suggest a novel role for BCL10 in the repair of DNA lesions.     

Blood and plasma titanium levels associated with well-functioning hip implants

BackgroundHip implants are usually manufactured from cobalt-chromium and titanium alloys. As the implants wear and corrode, metal debris is released into the surrounding tissue and blood, providing a potential biomarker for their function. Whilst there are laboratory reference levels for blood cobalt and chromium in patients with well and poorly functioning hip implants, there are no such guidelines for titanium. This is despite the increasing use of titanium implants worldwide.Patients and methodsWe recruited a consecutive series of 95 patients (mean age 71 years, mean time after surgery 8.5 years) with one hip implant type, inserted by the same surgeon. We assessed clinical and radiological outcome, and measured blood and plasma titanium using high resolution inductively-coupled plasma mass spectrometry.ResultsThe upper normal reference limit for blood and plasma titanium was 2.20 and 2.56 μg L⁻¹, respectively, and did not differ significantly between males and females.ConclusionWe are the first to propose a laboratory reference level for blood and plasma titanium in patients with well-functioning titanium hip implants. This is an essential starting point for further studies to explore the clinical usefulness of blood titanium as a biomarker of orthopaedic implant performance, and comes at a time of considerable controversy regarding the use of certain titanium alloys in hip arthroplasty.     

Hairless regulates p53 target genes to exert tumor suppressive functions in glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM-derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention.