The Folding Pathway of the Antibody VL Domain

Antibodies are modular proteins consisting of domains that exhibit a β-sandwich structure, the so-called immunoglobulin fold. Despite structural similarity, differences in folding and stability exist between different domains. In particular, the variable domain of the light chain V_L is unusual as it is associated with misfolding diseases, including the pathologic assembly of the protein into fibrillar structures. Here, we have analysed the folding pathway of a V_L domain with a view to determine features that may influence the relationship between productive folding and fibril formation. The V_L domain from MAK33 (murine monoclonal antibody of the subtype κ/IgG1) has not previously been associated with fibrillisation but is shown here to be capable of forming fibrils. The folding pathway of this V_L domain is complex, involving two intermediates in different pathways. An obligatory early molten globule-like intermediate with secondary structure but only loose tertiary interactions is inferred. The native state can then be formed directly from this intermediate in a phase that can be accelerated by the addition of prolyl isomerases. However, an alternative pathway involving a second, more native-like intermediate is also significantly populated. Thus, the protein can reach the native state via two distinct folding pathways. Comparisons to the folding pathways of other antibody domains reveal similarities in the folding pathways; however, in detail, the folding of the V_L domain is striking, with two intermediates populated on different branches of the folding pathway, one of which could provide an entry point for molecules diverted into the amyloid pathway.     

Birth weight,sex and childhood cancer: A report from the United Kingdom Childhood Cancer Study

Birth weight has been linked to the risk of developing childhood cancer, in particular childhood leukaemia. However, despite many childhood cancers having a male predominance and boys generally weighing more than girls at birth few studies have reported sex-specific associations. The relationship between birth weight and childhood cancer risk was examined using information from a national case-control study. Children (0–14 years) newly diagnosed with cancer in GB were ascertained between 1991 and 1996 (n = 3651) and for comparison, controls matched on sex, month and year of birth were identified from primary care population registers (n = 6337). Birth weights were obtained from the Office of National Statistics for all targeted subjects born in England and Wales. Overall, cases were, on average, 30 g heavier at birth than controls (p = 0.003) with differences seen by cancer type; those diagnosed with hepatic tumours weighing around 500 g less than controls at birth (p < 0.0001) and those with leukaemia being, on average, 50 g heavier than those without (p = 0.001). An interaction between birth weight and sex was found for acute leukaemia (χ² = 11.2, p = 0.04) and when data were stratified by sex, an association between high birth weight and risk of ALL was seen with girls (>4000 g, OR 1.86, 95% CI 1.38–2.50, χ² for trend 20.2, p < 0.0001). Our results support the hypothesis that birth weight is an important determinant for childhood cancer. In addition, the data are consistent with the notion that childhood leukaemia has a prenatal origin.     

Serum S100A6 Concentration Predicts Peritoneal Tumor Burden in Mice with Epithelial Ovarian Cancer and Is Associated with Advanced Stage in Patients

Background

Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival.

Methodology and Principal Findings

Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease.

Conclusions

S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers.     

Pneumocytes are distinguished by highly elevated expression of the ER stress biomarker GRP78, a co-receptor for SARS-CoV-2, in COVID-19 autopsies

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.     

FTIR determination of free fatty acids in fish oils intended for biodiesel production

Biodiesel is commonly derived from vegetable oils and animal (fish and livestock) fats by alkali- or lipase-catalyzed transesterification reactions. Since free fatty acid (FFA) content is a critical parameter in the conversion of fish oils to methyl esters, the performance of a Fourier transform infrared (FTIR) spectroscopic method was assessed as an alternative to the conventional AOCS titrimetric method. The FTIR method involves the simultaneous extraction of FFAs and their stoichiometric conversion to their salts using a weak base, sodium hydrogen cyanamide (NaHNCN) dissolved in methanol, followed by measurement of the carboxylate band, ν(COO^?), at 1573 cm^?1 relative to a baseline at 1820 cm^?1 in the differential spectrum of the methanol extract. With minor modifications, this method was found to be capable of responding linearly to oleic acid (0–6.5%) addition, producing a FFA calibration equation having a S.D. of ±0.014% FFA. FTIR and titrimetric analytical results were compared for samples prepared by standard addition as well as for fish oils extracted from salmon skin which had been stored up to 120 days at ?20 °C. Both methods responded in a comparable manner; however, the FTIR method was more reproducible and accurate as well as simpler to carry out and was deemed to be a better primary method than the titrimetric method. The FFA content of Atlantic salmon (Salmo salar) skin lipids increased linearly from ～0.6% to 4.5% within 120 days, likely as a result of autoxidation. It was concluded that the NaHNCN-based FTIR method is a flexible, viable instrumental alternative to the AOCS titrimetric procedure for the determination of FFA content of fish tissue lipids destined for biodiesel production.     

HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification

Over-expression and purification of soluble and functional proteins remain critical challenges for many aspects of biomolecular research. To address this, we have developed a novel protein tag, HaloTag7, engineered to enhance expression and solubility of recombinant proteins and to provide efficient protein purification coupled with tag removal. HaloTag7 was designed to bind rapidly and covalently with a unique synthetic linker to achieve an essentially irreversible attachment. The synthetic linker may be attached to a variety of entities such as fluorescent dyes and solid supports, permitting labeling of fusion proteins in cell lysates for expression screening, and efficient capture of fusion proteins onto a purification resin. The combination of covalent capture with rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional affinity tags and enables efficient capture even at low expression levels. Following immobilization on the resin, the protein of interest is released by cleavage at an optimized TEV protease recognition site, leaving HaloTag7 bound to the resin and pure protein in solution. Evaluation of HaloTag7 for expression of 23 human proteins in Escherichia coli relative to MBP, GST and His₆Tag revealed that 74% of the proteins were produced in soluble form when fused to HaloTag7 compared to 52%, 39% and 22%, respectively, for the other tags. Using a subset of the test panel, more proteins fused to HaloTag7 were successfully purified than with the other tags, and these proteins were of higher yield and purity.     

Biomarker assessment of organic matter sources and degradation in Canadian High Arctic littoral sediments

This paper presents an uncomplicated approach to improve estimates of groundwater nutrient load to a marine embayment. A two-dimensional chemical profile of shallow groundwater was analysed in a sandy beach in three seasons (early summer, late summer and mid winter) and an adjusted estimate of groundwater nutrient discharge was derived that accounts for a complex biogeochemical environment and non-conservative behaviour of nutrients in the pre-discharge beach groundwater. The study was conducted at Cockburn Sound, Western Australia, where there has been significant groundwater contamination and associated marine ecological degradation. Losses in nitrogen and increases in phosphorus were observed along the discharge pathway beyond that expected from mixing with marine water, and the changes were attributed to chemically and biologically mediated reactions. A slow groundwater velocity (0.14–0.18 m day^?1), high organic carbon (TOC = 0.35–4.9 mmol l^?1, DOC = 0.28–4.6 mmol l^?1) and low to sub-oxic conditions (DO = 0.4–24% saturation) were deemed suitable for chemically and biologically mediated reactions to occur and subsequently alter regional estimates of groundwater nutrient concentration. Accounting for this environment, groundwater loads were calculated that were 1–2 orders of magnitude less than previous regional-based estimates: 0.4–13 kg NO _x ^?  day^?1, 0.2–24 kg NH₄ ⁺ day^?1 and 0.004–0.8 kg FRP day^?1. This paper applies knowledge of recent research and presents scope to marine managers or modellers to account for groundwater inputs to the marine environment.     

Discovery of a series of indan carboxylic acid glycogen phosphorylase inhibitors

A series of carboxylic acid glycogen phosphorylase inhibitors, which have potential as oral antidiabetic agents, is described. Defining and applying simple physicochemical design criteria was used to assess the opportunity and to focus synthetic efforts on compounds with the greatest probability of success. The study led to compound 17, which exhibits a good balance of properties including potent inhibition of recombinant human liver glycogen phosphorylase in vitro, a good DMPK profile including excellent bioavailability and low clearance and good in vivo activity in a glucagon challenge model of diabetes in Zucker rats.     

Spirocyclic ureas: Orally bioavailable 11β-HSD1 inhibitors identified by computer-aided drug design

Structure-guided drug design led to the identification of a class of spirocyclic ureas which potently inhibit human 11β-HSD1 in vitro. Lead compound 10j was shown to be orally bioavailable in three species, distributed into adipose tissue in the mouse, and its (R) isomer 10j2 was efficacious in a primate pharmacodynamic model.