Molecular basis of the therapeutic properties of hemorphins.

Hemorphins are endogenous peptides, 4-10 amino acids long, belonging to the family of atypical opioid peptides released during the sequential cleavage of hemoglobin protein. Hemorphins have been shown to exhibit diverse therapeutic effects in both human and animal models. However, the precise cellular and molecular mechanisms involved in such effects remain elusive. In this review, we summarize and propose potential mechanisms based on studies that investigated the biological activity of hemorphins of different lengths on multiple therapeutic targets. Special emphasis is given to molecular events related to renin-angiotensin system (RAS), opioid receptors and insulin-regulated aminopeptidase receptor (IRAP). This review provides a comprehensive coverage of the molecular mechanisms that underpin the therapeutic potential of hemorphins. Furthermore, it highlights the role of various hemorphin residues in pathological conditions, which could be explored further for therapeutic purposes.

Impact of supervised exercise training on pulmonary function parameters, exercise capacity and Irisin Biomarker in Interstitial lung disease patients.

OBJECTIVES: To assess the impact of supervised exercise training (SET) on pulmonary function Parameters, exercise capacity and Irisin biomarker in Interstitial Lung Disease (ILD) patients. METHODS: Ten (10)patients with ILD and 18 healthy controls of age between 30-40+ years were selected for 8-week SET program. Before and after SET all subjects performed exercise capacity six minutes' walk test (6MWT), heart rate (HR) changes were recorded, shortness of Breath Respiratory Questionnaire (SOBQ) was obtained and Irisin levels were measured by Enzyme-Linked Immunosorbent Assay (ELISA). This interventional study was carried out at Department of Physiology, Faculty of Medicine, King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia, from October 2018 to February 2019. RESULTS: Mean six minutes' walk distance (6MWD) was 395 ± 68.4 m at 1st visit increased significantly (p=0.001) to 458.8± 87.1 mat 15 visit. However, 6MWD values found significantly higher in controls (517.4±84.1 m; 561.7±81.6 m; p=0.013) than ILD patients. Overall change (difference between post and pre exercise) in HRvalue was recorded lower in ILD patients (30-35 bpm) as compared to controls (40-45 bpm). Moreover, ILD patients had overall higher SOBQ score than controls. Pre SET Irisin levels of ILD patients (4.24 ±1.73 pg/ml) and controls (3.43 ±1.04pg/ml) were found unchanged dafter SET (4.48±2.02pg/ml, 3.39 ±1.41pg/ml, p=0.677, p=0.093)respectively. However, patients Irisin values were found higher as compared to controls before and after SET. CONCLUSION: Exercise capacity and Dyspneain patients with ILD were improved after 8-week of SET program. No major changes in Irisin levels among patients with ILD and controls were observed. Additional research requires to be carried out on large number of subjects to deter Minutese the advantages of exercise in ILD.

LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation.

Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that ∼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of ß-synuclein, PARK13, and SOD1 and causes ß-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.

Neurovirulence and immunogenicity of attenuated recombinant vesicular stomatitis viruses in nonhuman primates.

UNLABELLED: In previous work, a prototypic recombinant vesicular stomatitis virus Indiana serotype (rVSIV) vector expressing simian immunodeficiency virus (SIV) gag and human immunodeficiency virus type 1 (HIV-1) env antigens protected nonhuman primates (NHPs) from disease following challenge with an HIV-1/SIV recombinant (SHIV). However, when tested in a stringent NHP neurovirulence (NV) model, this vector was not adequately attenuated for clinical evaluation. For the work described here, the prototypic rVSIV vector was attenuated by combining specific G protein truncations with either N gene translocations or mutations (M33A and M51A) that ablate expression of subgenic M polypeptides, by incorporation of temperature-sensitive mutations in the N and L genes, and by deletion of the VSIV G gene to generate a replicon that is dependent on trans expression of G protein for in vitro propagation. When evaluated in a series of NHP NV studies, these attenuated rVSIV variants caused no clinical disease and demonstrated a very significant reduction in neuropathology compared to wild-type VSIV and the prototypic rVSIV vaccine vector. In spite of greatly increased in vivo attenuation, some of the rVSIV vectors elicited cell-mediated immune responses that were similar in magnitude to those induced by the much more virulent prototypic vector. These data demonstrate novel approaches to the rational attenuation of VSIV NV while retaining vector immunogenicity and have led to identification of an rVSIV N4CT1gag1 vaccine vector that has now successfully completed phase I clinical evaluation. IMPORTANCE: The work described in this article demonstrates a rational approach to the attenuation of vesicular stomatitis virus neurovirulence. The major attenuation strategy described here will be most likely applicable to other members of the Rhabdoviridae and possibly other families of nonsegmented negative-strand RNA viruses. These studies have also enabled the identification of an attenuated, replication-competent rVSIV vector that has successfully undergone its first clinical evaluation in humans. Therefore, these studies represent a major milestone in the development of attenuated rVSIV, and likely other vesiculoviruses, as a new vaccine platform(s) for use in humans.

PARK13 regulates PINK1 and subcellular relocation patterns under oxidative stress in neurons.

Parkinson's disease (PD) is a progressive and irreversible neurodegenerative disorder coupled to selective degeneration of dopamine-producing neurons in the substantia nigra. The majority of PD incidents are sporadic, but monogenic cases account for 5-10% of cases. Mutations in PINK1 cause autosomal recessive forms of early-onset PD, and PINK1 stimulates Omi/HtrA2/PARK13 protease activity when both proteins act as neuroprotective components in the same stress pathway. Studies on PINK1 and PARK13 have concentrated on phosphorylation-dependent PINK1-mediated activation of PARK13 and mitochondrial functions, because both proteins are classically viewed as mitochondrial. Although PARK13-mediated protective mechanisms are at least in part regulated by PINK1, little is known concerning how these two proteins are regulated in different subcellular compartments or, indeed, the influence of PARK13 on PINK1 characteristics. We show that PARK13 localizes to a variety of subcellular locations in neuronal cells and that PINK1, although more restrictive, also localizes to locations other than those previously reported. We demonstrate that PARK13 accumulation leads to a concomitant accumulation of PINK1 and that the increase in PINK1 levels is compartmental specific, indicating a correlative relationship between the two proteins. Moreover, we show that PARK13 and PINK1 protein levels accumulate in response to H2 O2 and L-DOPA treatments in a subcellular fashion and that both proteins show relocation to the cytoskeleton in response to H2 O2 . This H2 O2 -mediated relocation is abolished by PARK13 overexpression. This study shows that PARK13 and PINK1 are subcellular-specific, but dynamic, proteins with a reciprocal molecular relationship providing new insight into the complexity of PD.

Quality of Life, Fatigue, and Physical Symptoms Post-COVID-19 Condition: A Cross-Sectional Comparative Study.

The magnitude of post-COVID-19 syndrome was not thoroughly investigated. This study evaluated the quality of life and persistence of fatigue and physical symptoms of individuals post-COVID-19 compared with noninfected controls. The study included 965 participants; 400 had previous COVID-19 disease and 565 controls without COVID-19. The questionnaire collected data on comorbidities, COVID-19 vaccination, general health questions, and physical symptoms, in addition to validated measures of quality of life (SF-36 scale), fatigue (fatigue severity scale, FSS), and dyspnea grade. COVID-19 participants complained more frequently of weakness, muscle pain, respiratory symptoms, voice disorders, imbalance, taste and smell loss, and menstrual problems compared to the controls. Joint symptoms, tingling, numbness, hypo/hypertension, sexual dysfunction, headache, bowel, urinary, cardiac, and visual symptoms did not differ between groups. Dyspnea grade II-IV did not differ significantly between groups (p = 0.116). COVID-19 patients scored lower on the SF-36 domains of role physical (p = 0.045), vitality (p < 0.001), reported health changes (p < 0.001), and mental-components summary (p = 0.014). FSS scores were significantly higher in COVID-19 participants (3 (1.8-4.3) vs. 2.6 (1.4-4); p < 0.001). COVID-19 effects could persist beyond the acute infection phase. These effects include changes in quality of life, fatigue, and persistence of physical symptoms.

Accelerated antibody discovery targeting the SARS-CoV-2 spike protein for COVID-19 therapeutic potential.

BACKGROUND: Rapid deployment of technologies capable of high-throughput and high-resolution screening is imperative for timely response to viral outbreaks. Risk mitigation in the form of leveraging multiple advanced technologies further increases the likelihood of identifying efficacious treatments in aggressive timelines. METHODS: In this study, we describe two parallel, yet distinct, in vivo approaches for accelerated discovery of antibodies targeting the severe acute respiratory syndrome coronavirus-2 spike protein. Working with human transgenic Alloy-GK mice, we detail a single B-cell discovery workflow to directly interrogate antibodies secreted from plasma cells for binding specificity and ACE2 receptor blocking activity. Additionally, we describe a concurrent accelerated hybridoma-based workflow utilizing a DiversimAb™ mouse model for increased diversity. RESULTS: The panel of antibodies isolated from both workflows revealed binding to distinct epitopes with both blocking and non-blocking profiles. Sequence analysis of the resulting lead candidates uncovered additional diversity with the opportunity for straightforward engineering and affinity maturation. CONCLUSIONS: By combining in vivo models with advanced integration of screening and selection platforms, lead antibody candidates can be sequenced and fully characterized within one to three months.

Camel Hemorphins Exhibit a More Potent Angiotensin-I Converting Enzyme Inhibitory Activity than Other Mammalian Hemorphins: An In Silico and In Vitro Study.

Angiotensin-I converting enzyme (ACE) is a zinc metallopeptidase that has an important role in regulating the renin-angiotensin-aldosterone system (RAAS). It is also an important drug target for the management of cardiovascular diseases. Hemorphins are endogenous peptides that are produced by proteolytic cleavage of beta hemoglobin. A number of studies have reported various therapeutic activities of hemorphins. Previous reports have shown antihypertensive action of hemorphins via the inhibition of ACE. The sequence of hemorphins is highly conserved among mammals, except in camels, which harbors a unique Q>R variation in the peptide. Here, we studied the ACE inhibitory activity of camel hemorphins (LVVYPWTRRF and YPWTRRF) and non-camel hemorphins (LVVYPWTQRF and YPWTQRF). Computational methods were used to determine the most likely binding pose and binding affinity of both camel and non-camel hemorphins within the active site of ACE. Molecular dynamics simulations showed that the peptides interacted with critical residues in the active site of ACE. Notably, camel hemorphins showed higher binding affinity and sustained interactions with all three subsites of the ACE active site. An in vitro ACE inhibition assay showed that the IC50 of camel hemorphins were significantly lower than the IC50 of non-camel hemorphins.

Subcellular Parkinson's Disease-Specific Alpha-Synuclein Species Show Altered Behavior in Neurodegeneration.

Parkinson's disease and other synucleinopathies are characterized by the presence of intra-neuronal protein aggregates enriched in the presynaptic protein α-synuclein. α-synuclein is considered an intrinsically disordered 14 kDa monomer, and although poorly understood, its transition to higher-order multimeric species may play central roles in healthy neurons and during Parkinson's disease pathogenesis. In this study, we demonstrate that α-synuclein exists as defined, subcellular-specific species that change characteristics in response to oxidative stress in neuroblastoma cells and in response to Parkinson's disease pathogenesis in human cerebellum and frontal cortex. We further show that the phosphorylation patterns of different α-synuclein species are subcellular specific and dependent on the oxidative environment. Using high-performance liquid chromatography and mass spectrometry, we identify a Parkinson's disease enriched, cytosolic ~36-kDa α-synuclein species which can be recapitulated in Parkinson's disease model neuroblastoma cells. The characterization of subcellular-specific α-synuclein features in neurodegeneration will allow for the identification of neurotoxic α-synuclein species, which represent prime targets to reduce α-synuclein pathogenicity.

A fully human monoclonal antibody to the insulin-like growth factor I receptor blocks ligand-dependent signaling and inhibits human tumor growth in vivo.

The insulin-like growth factor I receptor (IGF-IR) is overexpressed in many diverse tumor types and is a critical signaling molecule for tumor cell proliferation and survival. Therapeutic strategies targeting the IGF-IR may therefore be effective broad-spectrum anticancer agents. Through screening of a Fab phage display library, we have generated a fully human antibody (A12) that binds to the IGF-IR with high affinity (4.11 x 10(-11) M) and inhibits ligand binding with an IC(50) of 0.6-1 nM. Antibody-mediated blockade of ligand binding to the IGF-IR inhibited downstream signaling of the two major insulin-like growth factor (IGF) pathways, mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/Akt, in MCF7 human breast cancer cells. As a result, the mitogenic and proliferative potential of IGF-I and IGF-II were significantly reduced. A12 did not block insulin binding to the insulin receptor but could block binding to atypical IGF-IR in MCF7 cells. In addition, A12 was shown to induce IGF-IR internalization and degradation on specific binding to tumor cells, resulting in a significant reduction in cell surface receptor density. In xenograft tumor models in vivo, IGF-IR blockade by A12 was shown to occur rapidly, resulting in significant growth inhibition of breast, renal, and pancreatic tumors. Histological analysis of tumor sections demonstrated a marked increase in apoptotic tumor cells in antibody-treated animals. These results demonstrate that A12 possesses strong antitumor activity in vitro and in vivo and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on IGF-IR signaling for growth and survival.

Parkinson's disease and age: The obvious but largely unexplored link.

Parkinson's disease is a chronic, progressive neurodegenerative disorder with increased prevalence in the aging population. It is estimated that approximately 1.5 million individuals in the US alone suffer from Parkinson's disease and with the extension of life expectancy this number is expected to rise dramatically within the next twenty-five years. The majority of Parkinson's disease cases are sporadic. But mutations in genes such as α-synuclein, Parkin, PINK1, DJ-1 and LRRK2, have been conclusively associated with both early- and late-onset of the disease. Although the genetics of Parkinson's disease is starting to become unraveled, the interplay between genetic and environmental factors is largely unknown as are the underlying mechanisms that trigger the disease as the brain ages. The risk of Parkinson's disease increases dramatically in individuals over the age of 60 and it is estimated that more than 1% of all seniors have some form of the condition. In this review, we will highlight some of the central proteins associated with Parkinson's disease and how they may be linked to processes and factors associated with age.

Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques.

Since human immunodeficiency virus (HIV)-specific cell-mediated immune (CMI) responses are critical in the early control and resolution of HIV infection and correlate with postchallenge outcomes in rhesus macaque challenge experiments, we sought to identify a plasmid DNA (pDNA) vaccine design capable of eliciting robust and balanced CMI responses to multiple HIV type 1 (HIV-1)-derived antigens for further development. Previously, a number of two-, three-, and four-vector pDNA vaccine designs were identified as capable of eliciting HIV-1 antigen-specific CMI responses in mice (M. A. Egan et al., Vaccine 24:4510-4523, 2006). We then sought to further characterize the relative immunogenicities of these two-, three-, and four-vector pDNA vaccine designs in nonhuman primates and to determine the extent to which in vivo electroporation (EP) could improve the resulting immune responses. The results indicated that a two-vector pDNA vaccine design elicited the most robust and balanced CMI response. In addition, vaccination in combination with in vivo EP led to a more rapid onset and enhanced vaccine-specific immune responses. In macaques immunized in combination with in vivo EP, we observed a 10- to 40-fold increase in HIV-specific enzyme-linked immunospot assay responses compared to those for macaques receiving a 5-fold higher dose of vaccine without in vivo EP. This increase in CMI responses translates to an apparent 50- to 200-fold increase in pDNA vaccine potency. Importantly, in vivo EP enhanced the immune response against the less immunogenic antigens, resulting in a more balanced immune response. In addition, in vivo EP resulted in an approximate 2.5-log(10) increase in antibody responses. The results further indicated that in vivo EP was associated with a significant reduction in pDNA persistence and did not result in an increase in pDNA associated with high-molecular-weight DNA relative to macaques receiving the pDNA without EP. Collectively, these results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

Alphavirus replicon particles encoding the fusion or attachment glycoproteins of respiratory syncytial virus elicit protective immune responses in BALB/c mice and functional serum antibodies in rhesus macaques.

Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract disease in humans. Towards development of a prophylactic vaccine, we genetically engineered Venezuelan equine encephalitis virus (VEEV) replicons encoding the fusion (Fa) or attachment (Ga or Gb) proteins of the A or B subgroups of RSV. Intramuscular immunization with a formulation composed of equal amounts of each replicon particle (3vRSV replicon vaccine) generated serum neutralizing antibodies against A and B strains of RSV in BALB/c mice and rhesus macaques. When contrasted with purified natural protein or formalin-inactivated RSV formulated with alum, the 3vRSV replicon vaccine induced balanced Th1/Th2 T cell responses in mice. This was evident in the increased number of RSV-specific IFN-gamma(+) splenocytes following F or G peptide stimulation, diminished quantity of eosinophils and type 2 T cell cytokines in the lungs after challenge, and increased in vivo lysis of RSV peptide-loaded target cells. The immune responses in mice were also protective against intranasal challenge with RSV. Thus, the replicon-based platform represents a promising new strategy for vaccines against RSV.

Identification of a transiently exposed VE-cadherin epitope that allows for specific targeting of an antibody to the tumor neovasculature.

VE-cadherin is an adhesion molecule localized at the adherens junctions of endothelial cells. It is crucial for the proper assembly of vascular structures during angiogenesis and maintaining vascular integrity. We have studied 3 monoclonal antibodies (mAbs) against murine VE-cadherin that inhibit angiogenesis and tumor growth. Two of these, BV13 and 10G4, also disrupted normal vessels, resulting in severe vascular leakage, whereas the third, E4G10, did not. The goal of the current report was to identify the epitope of E4G10 and distinguish it from those of the disruptive mAbs. We mapped the epitope of E4G10 to within the first 10 amino acids of mature VE-cadherin and demonstrated that conserved tryptophan residues in this sequence are required for VE-cadherin-mediated trans-adhesion. The disruptive mAbs target a different epitope within amino acids 45 to 56, which structural homology modeling suggests is not involved in trans-adhesion. From our studies, we hypothesize that E4G10 can only bind the neovasculature, where VE-cadherin has not yet engaged in trans-adhesion and its epitope is fully exposed. Thus, E4G10 can inhibit junction formation and angiogenesis but is unable to target normal vasculature because its epitope is masked. In contrast, BV13 and 10G4 bind an epitope that is accessible regardless of VE-cadherin interactions, leading to the disruption of adherens junctions. Our findings establish the immediate N-terminal region of VE-cadherin as a novel target for inhibiting angiogenesis.