A platform for efficient, thiol-stable conjugation to albumin's native single accessible cysteine

Herein we report the use of bromomaleimides for the construction of stable albumin conjugates via conjugation to its native, single accessible, cysteine followed by hydrolysis. Advantages over the classical maleimide approach are highlighted in terms of quantitative hydrolysis and absence of undesirable retro-Michael deconjugation

Human rights, non-refoulement and the protection of refugees in Hong Kong

Although the 1951 Convention relating to the Status of Refugees and its 1967 Protocol do not apply to Hong Kong, asylum seekers have challenged Hong Kong's lack of an adequate refugee policy in a series of judicial review actions grounded in human rights and common law principles. This article focuses on two cases in particular in which the applicants have attempted to rely, in part, on a right to non-refoulement, derived from international and domestic law, to compel the Government to establish procedures to determine the status of refugees and other similar categories of claimants. The first, Secretary for Security v. Sakthevel Prabakar, led to the creation of a 'torture screening' mechanism based on article 3 of the Convention against Torture and other Cruel, Inhuman, or Degrading Treatment or Punishment. In the second, C v. Director of Immigration, the court considered whether a rule of non-refoulement has emerged in customary international law and, if so, whether it applies to Hong Kong and requires government-administered refugee status determination. Although the applicants failed at first instance,1 an analysis of the judgment with reference to Hong Kong's human rights obligations reveals gaps in the court's reasoning and demonstrates the potential for greater reliance on these standards as the basis for developing a more comprehensive protection framework. This examination of the Hong Kong experience may have broader comparative value, especially in the Asian region and in jurisdictions not bound by the Refugee Convention or its Protocol. © The Author (2010). Published by Oxford University Press. All rights reserved.postprin

Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation.

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins

Advancing clinical and translational research in germ cell tumours (GCT): recommendations from the Malignant Germ Cell International Consortium

YesGerm cell tumours (GCTs) are a heterogeneous group of rare neoplasms that present in different anatomical sites and across a wide spectrum of patient ages from birth through to adulthood. Once these strata are applied, cohort numbers become modest, hindering inferences regarding management and therapeutic advances. Moreover, patients with GCTs are treated by different medical professionals including paediatric oncologists, neuro-oncologists, medical oncologists, neurosurgeons, gynaecological oncologists, surgeons, and urologists. Silos of care have thus formed, further hampering knowledge dissemination between specialists. Dedicated biobank specimen collection is therefore critical to foster continuous growth in our understanding of similarities and differences by age, gender, and site, particularly for rare cancers such as GCTs. Here, the Malignant Germ Cell International Consortium provides a framework to create a sustainable, global research infrastructure that facilitates acquisition of tissue and liquid biopsies together with matched clinical data sets that reflect the diversity of GCTs. Such an effort would create an invaluable repository of clinical and biological data which can underpin international collaborations that span professional boundaries, translate into clinical practice, and ultimately impact patient outcomes.ALF, JFA, and MJM declare funding from St Baldrick’s Foundation; grant reference number 358099

Over 1000 genetic loci influencing blood pressure with multiple systems and tissues implicated.

High blood pressure (BP) remains the major heritable and modifiable risk factor for cardiovascular disease. Persistent high BP, or hypertension, is a complex trait with both genetic and environmental interactions. Despite swift advances in genomics, translating new discoveries to further our understanding of the underlying molecular mechanisms remains a challenge. More than 500 loci implicated in the regulation of BP have been revealed by genome-wide association studies (GWAS) in 2018 alone, taking the total number of BP genetic loci to over 1000. Even with the large number of loci now associated to BP, the genetic variance explained by all loci together remains low (~5.7%). These genetic associations have elucidated mechanisms and pathways regulating BP, highlighting potential new therapeutic and drug repurposing targets. A large proportion of the BP loci were discovered and reported simultaneously by multiple research groups, creating a knowledge gap, where the reported loci to date have not been investigated in a harmonious way. Here, we review the BP-associated genetic variants reported across GWAS studies and investigate their potential impact on the biological systems using in silico enrichment analyses for pathways, tissues, gene ontology and genetic pleiotropy

Left gaze bias in humans, rhesus monkeys and domestic dogs

While viewing faces, human adults often demonstrate a natural gaze bias towards the left visual field, that is, the right side of the viewee’s face is often inspected first and for longer periods. Using a preferential looking paradigm, we demonstrate that this bias is neither uniquely human nor limited to primates, and provide evidence to help elucidate its biological function within a broader social cognitive framework. We observed that 6-month-old infants showed a wider tendency for left gaze preference towards objects and faces of different species and orientation, while in adults the bias appears only towards upright human faces. Rhesus monkeys showed a left gaze bias towards upright human and monkey faces, but not towards inverted faces. Domestic dogs, however, only demonstrated a left gaze bias towards human faces, but not towards monkey or dog faces, nor to inanimate object images. Our findings suggest that face- and species-sensitive gaze asymmetry is more widespread in the animal kingdom than previously recognised, is not constrained by attentional or scanning bias, and could be shaped by experience to develop adaptive behavioural significance

Surfactant protein D modulates HIV infection of both T-cells and dendritic cells

Surfactant Protein D (SP-D) is an oligomerized C-type lectin molecule with immunomodulatory properties and involvement in lung surfactant homeostasis in the respiratory tract. SP-D binds to the enveloped viruses, influenza A virus and respiratory syncytial virus and inhibits their replication in vitro and in vivo. SP-D has been shown to bind to HIV via the HIV envelope protein gp120 and inhibit infectivity in vitro. Here we show that SP-D binds to different strains of HIV (BaL and IIIB) and the binding occurs at both pH 7.4 and 5.0 resembling physiological relevant pH values found in the body and the female urogenital tract, respectively. The binding of SP-D to HIV particles and gp120 was inhibited by the presence of several hexoses with mannose found to be the strongest inhibitor. Competition studies showed that soluble CD4 and CVN did not interfere with the interaction between SP-D and gp120. However, soluble recombinant DC-SIGN was shown to inhibit the binding between SP-D and gp120. SP-D agglutinated HIV and gp120 in a calcium dependent manner. SP-D inhibited the infectivity of HIV strains at both pH values of 7.4 and 5.0 in a concentration dependent manner. The inhibition of the infectivity was abolished by the presence of mannose. SP-D enhanced the binding of HIV to immature monocyte derived dendritic cells (iMDDCs) and was also found to enhance HIV capture and transfer to the T-cell like line PM1. These results suggest that SP-D can bind to and inhibit direct infection of T-cells by HIV but also enhance the transfer of infectious HIV particles from DCs to T-cells in vivo

Structure and mechanism of human DNA polymerase η

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Pol eta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol eta in replicating through D loop and DNA fragile sites

Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria