Protein detection enabled using functionalised silk-binding peptides on a silk-coated optical fibre

We present a new coating procedure to prepare optical fibre sensors suitable for use with protein analytes. We demonstrate this through the detection of AlexaFluor-532 tagged streptavidin by its binding to D-biotin that is functionalised onto an optical fibre, via incorporation in a silk fibroin fibre coating. The D-biotin was covalently attached to a silk-binding peptide to provide SBP–biotin, which adheres the D-biotin to the silk-coated fibre tip. These optical fibre probes were prepared by two methods. The first involves dip-coating the fibre tip into a mixture of silk fibroin and SBP–biotin, which distributes the SBP–biotin throughout the silk coating (method A). The second method uses two steps, where the fibre is first dip-coated in silk only, then SBP–biotin added in a second dip-coating step. This isolates SBP–biotin to the outer surface of the silk layer (method B). A series of fluorescence measurements revealed that only the surface bound SBP–biotin detects streptavidin with a detection limit of 15 μg mL⁻¹. The fibre coatings are stable to repeated washing and long-term exposure to water. Formation of silk coatings on fibres using commercial aqueous silk fibroin was found to be inhibited by a lithium concentration of 200 ppm, as determined by atomic absorption spectroscopy. This was reduced to less than 20 ppm by dialysis against water, and was found to successfully form a coating on optical fibres.

We report a new approach to functionalise optical fibres to enable protein sensing, which controls the sensor molecule location either within the fibre tip coating or isolated to its exterior. This control dictates suitability for protein sensing.     

Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution

The newly emerging Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes a Severe Acute Respiratory Syndrome-like disease with ∼43% mortality. Given the recent detection of virus in dromedary camels, zoonotic transfer of MERS-CoV to humans is suspected. In addition, little is known about the role of human neutralizing Ab (nAb) pressure as a driving force in MERS-CoV adaptive evolution. Here, we used a well-characterized nonimmune human Ab-phage library and a panning strategy with proteoliposomes and cells to identify seven human nAbs against the receptor-binding domain (RBD) of the MERS-CoV Spike protein. These nAbs bind to three different epitopes in the RBD and human dipeptidyl peptidase 4 (hDPP4) interface with subnanomolar/nanomolar binding affinities and block the binding of MERS-CoV Spike protein with its hDPP4 receptor. Escape mutant assays identified five amino acid residues that are critical for neutralization escape. Despite the close proximity of the three epitopes on the RBD interface, escape from one epitope did not have a major impact on neutralization with Abs directed to a different epitope. Importantly, the majority of escape mutations had negative impacts on hDPP4 receptor binding and viral fitness. To our knowledge, these results provide the first report on human nAbs against MERS-CoV that may contribute to MERS-CoV clearance and evolution. Moreover, in the absence of a licensed vaccine or antiviral for MERS, this panel of nAbs offers the possibility of developing human mAb-based immunotherapy, especially for health-care workers.Middle East Respiratory Syndrome coronavirus (MERS-CoV), a newly emergent subgroup C betacoronavirus, was first isolated in the Arabian Peninsula in 2012 (1). Similar to the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) that emerged in China in 2002, MERS-CoV causes severe respiratory tract infection, often in the lower respiratory tract and occasionally accompanied by renal disease (1). As of February 28, 2014, 184 cases with 80 deaths have been confirmed in 10 countries in the Middle East, Europe, and North Africa (www.who.int/csr/don/2014_02_28/en/). Although the human-to-human transmission rate is mild to moderate at the moment (2, 3), the increasing number of person-to-person transmissions raises concern for a more widespread regional outbreak or even global spread by international travelers, as occurred with SARS-CoV in 2002–2003. Limited information exists on the mechanisms that confer increased human-to-human transmission of MERS-CoV (4). However, mutational adaptation of the SARS-CoV Spike (S) protein for its receptor, angiotensin-converting enzyme 2 (ACE2) was a positive selection factor after zoonotic transfer to humans (5, 6).Phylogenetic analysis indicates that MERS-CoV is closely related to CoVs detected in Tylonycteris pachypus and Pipistrellus abramus bats in China, Nyctinomops laticaudatus bats in Mexico, and Nycteris cf. gambiensis bats in Ghana and Europe (7–9). An ∼190-bp nucleotide fragment that was genetically identical to the RNA-dependent RNA polymerase of MERS-CoV was detected in Taphozous perforatus bat specimens in the vicinity of the index case in Saudi Arabia (10). Two independent serological surveys of livestock found that dromedary camels had a high prevalence of neutralizing Abs (nAbs) against MERS-CoV (11, 12). Recently, MERS-CoV has been identified from dromedary camels on a farm associated with two human cases, but the transmission patterns remain unclear (13). More recently, a study detected Abs in all 151 samples of dromedary camel serum obtained from the United Arab Emirates in 2003, indicating that MERS-CoV or closely related CoVs existed in the United Arab Emirates long before the first human MERS cases (14, 15). A screen of cell lines derived from livestock and peridomestic small mammals on the Arabian Peninsula revealed that only ungulates such as goats and camels showed efficient replication of MERS-CoV (16). These findings suggest that bats and camels may play an important role in MERS-CoV transmission and that the range of species that can be infected with MERS-CoV may be even broader than currently known (17).The coronavirus S protein is a class I membrane fusion protein that represents the major envelope protein on the surface of CoVs. The S protein presents as a trimer and mediates receptor binding, membrane fusion, and virus entry. S also is the major target for nAbs (18). It has been reported that patients infected with MERS-CoV generated S protein-specific nAbs (19, 20). The cellular receptor for MERS-CoV has been identified as dipeptidyl peptidase 4 (DPP4, CD26), which is conserved across many species (21). The receptor-binding domain (RBD) of the virus S protein in complex with human DPP4 (hDPP4) has been characterized (22, 23).Although MERS-CoV has a lower reproduction number (R₀) than SARS-CoV (0.69 vs. 0.80) (2), it has a much higher mortality rate (43% vs. 10%). Currently, no licensed vaccines or antivirals are available for the prevention or treatment of MERS. Combination treatment with IFN-α2b and ribavirin can moderate the host response and has been reported to improve clinical outcomes in MERS-CoV–infected rhesus macaques (24). MERS-CoV S protein vaccines based on modified vaccinia virus Ankara or Venezuelan Equine Encephalitis replicon particles and purified RBD can induce virus nAbs in mouse models (25–27). However, results of human studies have not been reported. Thus, an urgent medical need remains for the targeted prophylaxis and treatment of MERS.Human Ab engineering is a powerful tool that has been used for both discovery and therapeutic applications. We and others have proposed previously that human mAbs could be used in a outbreak setting for the prophylaxis and early treatment of emerging viral pathogens (28, 29). However, obtaining timely access to biological specimens from infected patients as a source of B cells for targeted selection or Ab-phage library construction is often challenging and can delay the discovery process (30–33). These restrictions have led us to use an ultra-large nonimmune human Ab-phage display library as a resource for the isolation of human nAbs to several emerging pathogens (29, 34, 35). With this Ab library resource and a unique panning strategy, we report the isolation and characterization of seven human nAbs that bind to three different epitopes at the MERS-CoV RBD–hDPP4 interface. We also investigated nAb-driven virus evolution and identified residues on the RBD that are critical for neutralization escape. These studies provide insight into the human nAb response that appears to impact MERS-CoV fitness and evolution. In addition, this panel of nAbs offers the possibility of developing a human mAb-based immunotherapy for the prevention and treatment of MERS.     

Durable Viral Suppression Among People with HIV and Problem Substance Use in the Era of Universal Antiretroviral Treatment

AIDS and Behavior - This study explored factors associated with durable viral suppression (DVS) among two groups of people living with HIV (PLWH) and problem substance use in the context of...     

Hypnotic Relaxation Therapy and Sexual Function in PostmenopausalWomen: Results of a Randomized Clinical Trial

Sexual dysfunction is a common problem for postmenopausal women. This study, as part of a larger randomized controlled trial, examined the effect of hypnotic relaxation therapy on sexual dysfunction, a secondary study outcome, in postmenopausal women. Sexual function was assessed using the Sexual Activity Questionnaire (SAQ). Significant improvement in sexual pleasure and discomfort were reported following 5 weekly sessions of hypnotic relaxation therapy, compared with those receiving an attention control. Total SAQ scores showed significant improvement in the hypnotic relaxation therapy treatment group while holding baseline SAQ scores constant. Improvements showed a slight increase at the Week 12 follow-up. The results of this analysis provide initial support for the use of hypnotic relaxation therapy to improve sexual function in postmenopausal women.     

B cell receptor signaling in human systemic lupus erythematosus

PURPOSE OF REVIEW: The purpose of this review is to inform the scientific community of the most recent findings surrounding B cell receptor signaling function in human systemic lupus erythematosus and how altered B cell signaling may explain the characteristic hyperactivity of B cells in active disease and contribute to its pathogenesis. RECENT FINDINGS: B cell receptor signaling is abnormal in patients with active systemic lupus erythematosus as demonstrated by increased calcium flux and global B cell hyperactivity. Altered signaling has been explained by a variety of factors such as defective FcgammaRIIB signaling, decreased expression of the protein tyrosine kinase Lyn, and increased serum levels of B lymphocyte stimulator. SUMMARY: The studies reviewed suggest that B cells from systemic lupus erythematosus patients display molecular signaling defects that most likely contribute to pathogenesis of the disease and explain the characteristic hyperactivity of B cells in active disease.     

Undiagnosed Primary Hyperparathyroidism and Recurrent Miscarriage: The First Prospective Pilot Study

Background

Primary hyperparathyroidism (pHPT) in pregnancy is reported to be associated with significant maternal and foetal complications and an up to threefold increase in the risk of miscarriage. However, the true incidence of pHPT in pregnancy, complete and miscarried, is unknown and there are no data on the prevalence of undiagnosed pHPT in recurrent miscarriage (RM) (≥3 consecutive miscarriages under 24-week gestation). This is the first prospective study aiming to establish the prevalence of undiagnosed pHPT in RM.

Methods

Following UK National ethics committee approval, women who had experienced 3 or more consecutive miscarriages were recruited from a nationwide RM clinic. Serum corrected calcium, phosphate, PTH and vitamin D were evaluated. Patients with raised serum calcium and/or PTH were recalled for confirmatory tests. Power calculations suggested that a minimum of 272 patients were required to demonstrate a clinically significant incidence of pHPT.

Results

Three hundred women were recruited, median age 35 years (range 19–42). Eleven patients had incomplete data, leaving 289 patients suitable for analysis; 50/289 patients (17%) with abnormal tests were recalled. The prevalence of vitamin D deficiency (<25 nmol/l) and insufficiency (25–75 nmol/l) was 8.7 and 67.8%, respectively. One patient was diagnosed with pHPT (0.34%) and underwent successful parathyroidectomy.

Conclusions

The prevalence of undiagnosed pHPT (0.34%) in RM in this study appears to be many times greater than the 0.05% expected in this age group. The findings of this pilot study merit follow-up with a larger-scale study. Routine serum calcium estimation is not currently undertaken in RM and should be considered.

     

Peripheral artery disease, diabetes, and reduced lower extremity functioning.

OBJECTIVE: To characterize lower extremity function and dysfunction in peripheral artery disease (PAD) patients with and without diabetes. RESEARCH DESIGN AND METHODS: In this cross-sectional study, 460 men and women with PAD (147 with diabetes) were recruited from three academic medical centers. Assessments included ankle brachial index (ABI), neuropathy score, 6-min walk distance, 4-m walking velocity, Walking Impairment Questionnaire (0-100 scale, 100 = best), and summary performance score (SPS) (0-12 scale, 12 = best). RESULTS: The mean ABI was similar in PAD patients with and without diabetes. PAD patients with diabetes were younger, had a higher BMI, had a worse neuropathy score, and had a greater number of cardiovascular comorbidities compared with those without diabetes. Participants with diabetes were less likely to report classical symptoms of intermittent claudication and more likely to report exertional leg pain, which sometimes started at rest. After adjusting for age, those with diabetes had a shorter mean 6-min walk distance (1,040 vs. 1,168 feet, P < 0.001), slower fast-pace 4-m walk velocity (0.83 vs. 0.90 m/sec, P < 0.001), and a lower SPS (7.3 vs. 8.6, P < 0.001) than those without diabetes. Patients with diet-controlled diabetes performed better than those on diabetes medications. Differences in lower extremity functioning between patients with and without diabetes were largely attenuated but not abolished for SPS and fast-pace 4-m walk velocity after adjustment for type of exertional leg pain, neuropathy score, and number of cardiovascular comorbidities. CONCLUSIONS: Subjects with PAD and diabetes have poorer lower extremity function than those with PAD alone. This difference in functioning appears to be largely explained by diabetes-associated neuropathy, differences in exertional leg symptoms, and greater cardiovascular disease in patients with diabetes.     

Expert opinions on success factors for upper-limb prostheses

The goal of this study was to gather the opinions of prosthetics experts on the most important factors for the successful use of upper-limb (UL) prostheses, compare them with those of prosthesis users, and ultimately direct research efforts in this field. UL prosthetics experts were asked to compare the importance of the comfort, function, and cosmesis of a prosthetic device for a transhumeral amputee. Categories were subdivided into weight, socket-interface comfort, power, agility, color, and shape. The majority of those who responded viewed comfort as the most important factor for a unilateral amputee and considered socket-interface comfort to be more important than weight. Function was considered to be the most important factor for a bilateral amputee, with agility considered more important than power. Cosmesis was consistently reported as being less important than comfort and function, and shape was considered more important than color.