Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV

Chronic Intranasal Treatment with an Anti-Aβ30-42 scFv Antibody Ameliorates Amyloid Pathology in a Transgenic Mouse Model of Alzheimer's Disease

Amyloid-beta peptide (Aβ)-directed active and passive immunization therapeutic strategies reduce brain levels of Aβ, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aβ-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aβ, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aβ-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aβ fibril formation and Aβ-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aβ from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aβ-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aβ-directed immunotherapy can reduce Aβ deposition in brain vessels in transgenic mice

Differential Cytoskeletal Responses to ATP Depletion and Repletion in Renal Proximal Tubule and Interstitial Fibroblast Cells

Acute kidney injury (AKI) is a growing problem in the United States. The high morbidity and mortality of this disease combined with limited treatment options make AKI an important area of research. Renal proximal tubule and interstitial fibroblast cells respond differently to AKI. Actin cytoskeleton structure in proximal tubule cells is disrupted during an ischemic event (Kellerman and Bogusky, 1992). This change is mediated by cofilin activation and altered tropomyosin localization (Schwartz et al., 1999, Ashworth et al., 2001, Ashworth et al., 2004). While changes in cofilin and tropomyosin have been identified during ischemia, the role of these proteins in recovery is not yet fully understood. The presence of interstitial fibroblasts and extracellular matrix proteins is increased after an ischemic event (Forbes et al., 2000). This increase can lead to a condition known as fibrosis, which leads to end stage renal failure (Qi et al., 2006). The role of cofilin and tropomyosin in this process remains unclear. In this study, we observed increased cofilin activation in proximal tubule and interstitial fibroblast cells with twenty minutes of ATP depletion, a model for renal ischemia. With twenty-four hours of ATP repletion, cofilin activation was decreased in interstitial fibroblast cells but increased in proximal tubule cells. At this time point actin stress fibers and cell-to-cell contacts were disrupted in proximal tubule cells, while tropomyosin bound stress fibers were present in renal interstitial fibroblasts. These findings suggested that the differential response of proximal tubule cells and interstitial fibroblasts to AKI may be a result of differences in cofilin activation and tropomyosin localization in these cells. The death-associated protein kinase (DAPK) family proteins are regulators of the actin cytoskeleton (Bialik and Kimchi, 2006). DAPK1 is activated during recovery from ischemia of the brain, leading to its degradation (Shamloo et al., 2005, Schumacher et al., 2002). DAPK2 expression and localization have not been examined in renal ischemia and may signal to the actin cytoskeleton during or after AKI. These studies demonstrated that with twenty-four hours of recovery from ATP depletion, DAPK2 expression was decreased in proximal tubule cells, but increased in renal interstitial fibroblasts. These differences in DAPK2 expression provided hints to a possible mechanism leading to the observed changes in the actin cytoskeleton during recovery from ATP depletion. Understanding the differential responses of proximal tubule cells and renal interstitial fibroblasts during AKI may lead to better treatment options for this disease. Interruption of signals that lead to proximal tubule disruption and fibroblast activation during and after AKI may reduce the incidence of end stage renal failure that can accompany AKI

Rapid generation of potent antibodies by autonomous hypermutation in yeast

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe 'autonomous hypermutation yeast surface display' (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large

Are total, intensity- and domain-specific physical activity levels associated with life satisfaction among university students?

BACKGROUND: Thorough information about the relationship between physical activity (PA) and life satisfaction is still lacking. Therefore, this study examined the cross-sectional relationships between life satisfaction and meeting the World Health Organization (WHO) moderate to vigorous-intensity PA recommendations, total volume and duration of PA, intensity-specific PA (walking, moderate- and vigorous-intensity), domain-specific PA (work, transport-related, domestic, and leisure-time), and 11 domain and intensity-specific PA types among university students. Additionally, we examined the associations between life satisfaction and gender, age, disposable income, community size, smoking, alcohol intake, body mass index (BMI), and self-rated health. METHODS: The study included a random sample of 1750 university students in Zagreb, Croatia (response rate = 71.7%; 62.4% females; mean age 21.5 ± 1.8 years), using the International Physical Activity Questionnaire-long form and the Satisfaction with Life Scale. RESULTS: Higher life satisfaction was associated with female gender (β = 0.13; p = <0.001), younger age (β = -0.07; p = 0.024), higher disposable income (β = 0.10; p = 0.001), and better self-rated health (β = 0.30; p = <0.001). No significant association was found between life satisfaction and size of community (p = 0.567), smoking status (p = 0.056), alcohol consumption (p = 0.058), or BMI (p = 0.508). Among all PA variables, only leisure-time vigorous-intensity PA was significantly associated with life satisfaction after adjustments for socio-demographic characteristics, lifestyle and self-rated general health (β = 0.06; p = 0.045). CONCLUSIONS: This study indicated a weak positive relationship between leisure-time vigorous-intensity PA and life satisfaction, whilst no such association was found for other PA variables. These findings underscore the importance of analyzing domain and intensity-specific PA levels in future studies among university students, as drawing conclusions about the relationship between PA and life satisfaction based on total PA levels only may be misleading