CyDisCo production of functional recombinant SARS‐CoV‐2 spike receptor binding domain

The COVID‐19 pandemic caused by SARS‐CoV‐2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of a SARS‐CoV‐2 spike protein receptor binding domain (RBD) that includes the SD1 domain. Bacterial cytoplasm is a reductive environment, which is problematic when the recombinant protein of interest requires complicated folding and/or processing. The use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) bypasses this issue by pre‐expressing a sulfhydryl oxidase and a disulfide isomerase, allowing the recombinant protein to be correctly folded with disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce an active RBD in bacteria that is capable of recognizing and binding to the ACE2 (angiotensin‐converting enzyme) receptor as well as antibodies in COVID‐19 patient sera.     

Resilience of inshore, juvenile snapper Pagrus auratus to angling and release

This study assessed the mortality of 157 snapper Pagrus auratus (9-29 cm, total length, L(T) ) after being conventionally angled and then released into cages (along with 48 controls) for 4 days off south-eastern Australia. Fatalities were restricted to 12 angled fish (7·6%) and mostly attributed to the ingestion of hooks and especially their subsequent removal, which caused substantial blood loss and immediate death. Hook ingestion was significantly biased towards smaller fish (<21 cm L(T)) and attributed to a lower chance of anglers initially detecting these individuals on the line (allowing them to consume more of the baits). While mortalities might be reduced in future via (1) choosing terminal rigs that promote mouth hooking and (2) cutting the line on any-hook ingested fish, the results nevertheless validate releasing unwanted angled inshore juvenile P. auratus as a means for managing their exploitation.     

Helminthic therapy: improving mucosal barrier function

The epidemiology of autoimmune diseases and helminth infections led to suggestions that helminths could improve inflammatory conditions, which was then tested using animal models. This has translated to clinical investigations aimed at the safe and controlled reintroduction of helminthic exposure to patients suffering from autoimmune diseases (so-called 'helminthic therapy') in an effort to mitigate the inflammatory response. In this review, we summarize the results of recent clinical trials of helminthic therapy, with particular attention to mechanisms of action. Whereas previous reviews have emphasized immune regulatory mechanisms activated by helminths, we propose that enhancement of mucosal barrier function may have an equally important role in improving conditions of inflammatory bowel diseases.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

Metabolic profiling of serum using Ultra Performance Liquid Chromatography and the LTQ-Orbitrap mass spectrometry system

Advances in analytical instrumentation can provide significant advantages to the volume and quality of biological knowledge acquired in metabolomic investigations. The interfacing of sub-2mum liquid chromatography (UPLC ACQUITY((R))) and LTQ-Orbitrap mass spectrometry systems provides many theoretical advantages. The applicability of the interfaced systems was investigated using a simple 11-component metabolite mix and a complex mammalian biofluid, serum. Metabolites were detected in the metabolite mix with signals that were linear with their concentration over 2.5-3.5 orders of magnitude, with correlation coefficients greater than 0.993 and limits of detection less than 1mumolL(-1). Reproducibility of retention time (RSD<3%) and chromatographic peak area (RSD<15%) and a high mass accuracy (<2ppm) were observed for 14 QC serum samples interdispersed with other serum samples, analysed over a period of 40h. The evaluation of a single deconvolution software package (XCMS) was performed and showed that two parameters (snthresh and bw) provided significant changes to the number of peaks detected and the peak area reproducibility for the dataset used. The data were used to indicate possible biomarkers of pre-eclampsia and showed both the instruments and XCMS to be applicable to the reproducible and valid detection of disease biomarkers present in serum.     

Mortality of discards from southeastern Australian beach seines and gillnets

Two experiments were done in an Australian estuary to quantify the mortalities and contributing factors for key species discarded during 8 and 9 deployments of commercial beach (or shore) seines and gillnets, respectively. In both experiments, bycatches (2347 individuals comprising 16 species) were handled according to conventional practices and assessed for immediate mortalities before live samples of selected species were discarded into replicate cages along with appropriate controls, and monitored for short-term mortalities (< or =10 d). All of the seined or gilled fish were alive prior to discarding. During the beach seine experiment, 20% of caged seined-and-discarded surf bream Acanthopagrus australis (n = 290) were dead after 5 d, with most mortalities occurring between the second and fifth day. In the gillnet experiment, 42 and 11% of gilled-and-discarded A. australis (n = 161) and lesser salmon catfish Neoarius graeffei (n = 67), respectively, died during a 10 d monitoring period, mostly within the first 5 d. There were no deaths in any controls for these fish. Mixed-effects logistic models revealed that the mortality of A. australis discarded from both gears was significantly (p < 0.01) and negatively correlated with their total length, while N. graeffei had a significantly (p < 0.05) greater (5-fold) probability of dying when jellyfish Catostylus sp. were present in the gillnet. Simple modifications to the operations of beach seines and gillnets and/or post-capture handling procedures, such as close regulation of size selectivity for the target species, careful removal of fish from meshes, and abstention from setting during high abundances of jellyfish will maximise the survival of discarded bycatch.     

Use of the EM algorithm to detect QTL affecting multiple-traits in an across half-sib family analysis

QTL detection experiments in livestock species commonly use the half-sib design. Each male is mated to a number of females, each female producing a limited number of progeny. Analysis consists of attempting to detect associations between phenotype and genotype measured on the progeny. When family sizes are limiting experimenters may wish to incorporate as much information as possible into a single analysis. However, combining information across sires is problematic because of incomplete linkage disequilibrium between the markers and the QTL in the population. This study describes formulæ for obtaining MLEs via the expectation maximization (EM) algorithm for use in a multiple-trait, multiple-family analysis. A model specifying a QTL with only two alleles, and a common within sire error variance is assumed. Compared to single-family analyses, power can be improved up to fourfold with multi-family analyses. The accuracy and precision of QTL location estimates are also substantially improved. With small family sizes, the multi-family, multi-trait analyses reduce substantially, but not totally remove, biases in QTL effect estimates. In situations where multiple QTL alleles are segregating the multi-family analysis will average out the effects of the different QTL alleles.