Molecular dynamics simulation study on the structures of fascin mutants.

Fascin is a filamentous actin (F-actin) bundling protein, which cross-links F-actin into bundles and becomes an important component of filopodia on the cell surface. Fascin is overexpressed in many types of cancers. The mutation of fascin affects its ability to bind to F-actin and the progress of cancer. In this paper, we have studied the effects of residues of K22, K41, K43, K241, K358, K399, and K471 using molecular dynamics (MD) simulation. For the strong-effect residues, that is, K22, K41, K43, K358, and K471, our results show that the mutation of K to A leads to large values of root mean square fluctuation (RMSF) around the mutated residues, indicating those residues are important for the flexibility and thermal stability. On the other hand, based on residue cross-correlation analysis, alanine mutations of these residues reinforce the correlation between residues. Together with the RMSF data, the local flexibility is extended to the entire protein by the strong correlations to influence the dynamics and function of fascin. By contrast, for the mutants of K241A and K399A those do not affect the function of fascin, the RMSF data do not show significant differences compared with wild-type fascin. These findings are in a good agreement with experimental studies.

Rational design of an anti-cancer peptide inhibiting CD147 / Cyp A interaction.

The CD147 / Cyp A interaction is a critical pathway in cancer types and an essential factor in entering the COVID-19 virus into the host cell. Melittin acts as an inhibitory peptide in cancer types by blocking the CD147/ Cyp A interaction. The clinical application of Melittin is limited due to weak penetration into cancer cells. TAT is an arginine-rich peptide with high penetration ability into cells widely used in drug delivery systems. This study aimed to design a hybrid peptide derived from Melittin and TAT to inhibit CD147 /Cyp A interaction. An amino acid region with high anti-cancer activity in Melittin was selected based on the physicochemical properties. Based on the results, a truncated Melittin peptide with 15 amino acids by the GGGS linker was fused to a TAT peptide (nine amino acids) to increase the penetration rate into the cell. A new hybrid peptide analog(TM) was selected by replacing the glycine with serine based on random point mutation. Docking results indicated that the TM peptide acts as an inhibitory peptide with high binding energy when interacting with CD147 and the CypA proteins. RMSD and RMSF results confirmed the high stability of the TM peptide in interaction with CD147. Also, the coarse-grained simulation showed the penetration potential of TM peptide into the DOPS-DOPC model membrane. Our findings indicated that the designed multifunctional peptide could be an attractive therapeutic candidate to halter tumor types and COVID-19 infection.

Glycyrrhizin as a promising kryptonite against SARS-CoV-2: Clinical, experimental, and theoretical evidences.

COVID-19 is the most devastating disease in recent times affecting most people globally. The higher rate of transmissibility and mutations of SARS-CoV-2 along with the lack of potential therapeutics has made it a global crisis. Potential molecules from natural sources could be a fruitful remedy to combat COVID-19. This systematic review highlights the detailed therapeutic implication of naturally occurring glycyrrhizin and its related derivatives against COVID-19. Glycyrrhizin has already been established for blocking different biomolecular targets related to the SARS-CoV-2 replication cycle. In this article, several experimental and theoretical evidences of glycyrrhizin and related derivatives have been discussed in detail to evaluate their potential as a promising therapeutic strategy against COVID-19. Moreover, the implication of glycyrrhizin in traditional Chinese medicines for alleviating the symptoms of COVID-19 has been reviewed. The potential role of glycyrrhizin and related compounds in affecting various stages of the SARS-CoV-2 life cycle has also been discussed in detail. Derivatization of glycyrrhizin for designing potential lead compounds along with combination therapy with other anti-SARS-CoV-2 agents followed by extensive evaluation may assist in the formulation of novel anti-coronaviral therapy for better treatment to combat COVID-19.

Interactive deciphering electron-shuttling characteristics of Coffea arabica leaves and potential bioenergy-steered anti-SARS-CoV-2 RdRp inhibitor via microbial fuel cells.

Due to the pandemics of COVID-19, herbal medicine has recently been explored for possible antiviral treatment and prevention via novel platform of microbial fuel cells. It was revealed that Coffea arabica leaves was very appropriate for anti-COVID-19 drug development. Antioxidant and anti-inflammatory tests exhibited the most promising activities for C. arabica ethanol extracts and drying approaches were implemented on the leaf samples prior to ethanol extraction. Ethanol extracts of C. arabica leaves were applied to bioenergy evaluation via DC-MFCs, clearly revealing that air-dried leaves (CA-A-EtOH) exhibited the highest bioenergy-stimulating capabilities (ca. 2.72 fold of power amplification to the blank). Furthermore, molecular docking analysis was implemented to decipher the potential of C. arabica leaves metabolites. Chlorogenic acid (-6.5 kcal/mol) owned the highest binding affinity with RdRp of SARS-CoV-2, showing a much lower average RMSF value than an apoprotein. This study suggested C. arabica leaves as an encouraging medicinal herb against SARS-CoV-2.

Multi-ligand molecular docking, simulation, free energy calculations and wavelet analysis of the synergistic effects between natural compounds baicalein and cubebin for the inhibition of the main protease of SARS-CoV-2.

Combination drugs have been used for several diseases for many years since they produce better therapeutic effects. However, it is still a challenge to discover candidates to form a combination drug. This study aimed to investigate whether using a comprehensive in silico approach to identify novel combination drugs from a Chinese herbal formula is an appropriate and creative strategy. We, therefore, used Toujie Quwen Granules for the main protease (Mpro) of SARS-CoV-2 as an example. We first used molecular docking to identify molecular components of the formula which may inhibit Mpro. Baicalein (HQA004) is the most favorable inhibitory ligand. We also identified a ligand from the other component, cubebin (CHA008), which may act to support the proposed HQA004 inhibitor. Molecular dynamics simulations were then performed to further elucidate the possible mechanism of inhibition by HQA004 and synergistic bioactivity conferred by CHA008. HQA004 bound strongly at the active site and that CHA008 enhanced the contacts between HQA004 and Mpro. However, CHA008 also dynamically interacted at multiple sites, and continued to enhance the stability of HQA004 despite diffusion to a distant site. We proposed that HQA004 acted as a possible inhibitor, and CHA008 served to enhance its effects via allosteric effects at two sites. Additionally, our novel wavelet analysis showed that as a result of CHA008 binding, the dynamics and structure of Mpro were observed to have more subtle changes, demonstrating that the inter-residue contacts within Mpro were disrupted by the synergistic ligand. This work highlighted the molecular mechanism of synergistic effects between different herbs as a result of allosteric crosstalk between two ligands at a protein target, as well as revealed that using the multi-ligand molecular docking, simulation, free energy calculations and wavelet analysis to discover novel combination drugs from a Chinese herbal remedy is an innovative pathway.

Screening and identification of potential MERS-CoV papain-like protease (PLpro) inhibitors; Steady-state kinetic and Molecular dynamic studies.

MERS-CoV belongs to the coronavirus group. Recent years have seen a rash of coronavirus epidemics. In June 2012, MERS-CoV was discovered in the Kingdom of Saudi Arabia, with 2,591 MERSA cases confirmed by lab tests by the end of August 2022 and 894 deaths at a case-fatality ratio (CFR) of 34.5% documented worldwide. Saudi Arabia reported the majority of these cases, with 2,184 cases and 813 deaths (CFR: 37.2%), necessitating a thorough understanding of the molecular machinery of MERS-CoV. To develop antiviral medicines, illustrative investigation of the protein in coronavirus subunits are required to increase our understanding of the subject. In this study, recombinant expression and purification of MERS-CoV (PLpro), a primary goal for the development of 22 new inhibitors, were completed using a high throughput screening methodology that employed fragment-based libraries in conjunction with structure-based virtual screening. Compounds 2, 7, and 20, showed significant biological activity. Moreover, a docking analysis revealed that the three compounds had favorable binding mood and binding free energy. Molecular dynamic simulation demonstrated the stability of compound 2 (2-((Benzimidazol-2-yl) thio)-1-arylethan-1-ones) the strongest inhibitory activity against the PLpro enzyme. In addition, disubstitutions at the meta and para locations are the only substitutions that may boost the inhibitory action against PLpro. Compound 2 was chosen as a MERS-CoV PLpro inhibitor after passing absorption, distribution, metabolism, and excretion studies; however, further investigations are required.

Structure-based virtual screening, in silico docking, ADME properties prediction and molecular dynamics studies for the identification of potential inhibitors against SARS-CoV-2 Mpro.

COVID-19 is a viral pandemic caused by SARS-CoV-2. Due to its highly contagious nature, millions of people are getting affected worldwide knocking down the delicate global socio-economic equilibrium. According to the World Health Organization, COVID-19 has affected over 186 million people with a mortality of around 4 million as of July 09, 2021. Currently, there are few therapeutic options available for COVID-19 control. The rapid mutations in SARS-CoV-2 genome and development of new virulent strains with increased infection and mortality among COVID-19 patients, there is a great need to discover more potential drugs for SARS-CoV-2 on a priority basis. One of the key viral enzymes responsible for the replication and maturation of SARS-CoV-2 is Mpro protein. In the current study, structure-based virtual screening was used to identify four potential ligands against SARS-CoV-2 Mpro from a set of 8,722 ASINEX library compounds. These four compounds were evaluated using ADME filter to check their ADME profile and druggability, and all the four compounds were found to be within the current pharmacological acceptable range. They were individually docked to SARS-CoV-2 Mpro protein to assess their molecular interactions. Further, molecular dynamics (MD) simulations was carried out on protein-ligand complex using Desmond at 100 ns to explore their binding conformational stability. Based on RMSD, RMSF and hydrogen bond interactions, it was found that the stability of protein-ligand complex was maintained throughout the entire 100 ns simulations for all the four compounds. Some of the key ligand amino acid residues participated in stabilizing the protein-ligand interactions includes GLN 189, SER 10, GLU 166, ASN 142 with PHE 66 and TRP 132 of SARS-CoV-2 Mpro. Further optimization of these compounds could lead to promising drug candidates for SARS-CoV-2 Mpro target.

Isolate-Dependent Differences in Clinical, Pathological, and Transcriptional Profiles following In Vitro and In Vivo Infections with Rickettsia rickettsii.

Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever (RMSF), a life-threatening tick-borne disease that affects humans and various animal species, has been recognized in medicine and science for more than 100 years. Isolate-dependent differences in virulence of R. rickettsii have been documented for many decades; nonetheless, the specific genetic and phenotypic factors responsible for these differences have not been characterized. Using in vivo and in vitro methods, we identified multiple phenotypic differences among six geographically distinct isolates of R. rickettsii, representing isolates from the United States, Costa Rica, and Brazil. Aggregate phenotypic data, derived from growth in Vero E6 cells and from clinical and pathological characteristics following infection of male guinea pigs (Cavia porcellus), allowed separation of these isolates into three categories: nonvirulent (Iowa), mildly virulent (Sawtooth and Gila), and highly virulent (Sheila SmithT, Costa Rica, and Taiaçu). Transcriptional profiles of 11 recognized or putative virulence factors confirmed the isolate-dependent differences between mildly and highly virulent isolates. These data corroborate previous qualitative assessments of strain virulence and suggest further that a critical and previously underappreciated balance between bacterial growth and host immune response could leverage strain pathogenicity. Also, this work provides insight into isolate-specific microbiological factors that contribute to the outcome of RMSF and confirms the hypothesis that distinct rickettsial isolates also differ phenotypically, which could influence the severity of disease in vertebrate hosts.

Identification of alkaloids from Justicia adhatoda as potent SARS CoV-2 main protease inhibitors: An in silico perspective.

The COVID-19 pandemic, caused by SARS CoV-2, is responsible for millions of death worldwide. No approved/proper therapeutics is currently available which can effectively combat this outbreak. Several attempts have been undertaken in the search of effective drugs to control the spread of SARS CoV-2 infection. The main protease (Mpro), key component for the cleavage of the viral polyprotein, is considered to be one of the important drug targets for treating COVID-19. Various phytochemicals, including polyphenols and alkaloids, have been proposed as potent inhibitors of Mpro. The alkaloids from leaf extracts of Justicia adhatoda have also been reported to possess anti-viral activity. But whether these alkaloids exhibit any inhibitory effect on SARS CoV-2 Mpro is far from clear. To explore this in detail, we have adopted computational approaches. Justicia adhatoda alkaloids possessing proper drug-likeness properties and two anti-HIV drugs (lopinavir and darunavir; having binding affinity -7.3 to -7.4 kcal/mol) were docked against SARS CoV-2 Mpro to study their binding properties. Only one alkaloid (anisotine) had interaction with both the catalytic residues (His41 and Cys145) of Mpro and exhibited good binding affinity (-7.9 kcal/mol). Molecular dynamic simulations (100 ns) revealed that Mpro-anisotine complex is more stable, conformationally less fluctuated; slightly less compact and marginally expanded than Mpro-darunavir/lopinavir complex. Even the number of intermolecular H-bonds and MM-GBSA analysis suggested that anisotine is a more potent Mpro inhibitor than the two previously recommended antiviral drugs (lopinavir and darunavir) and may evolve as a promising anti-COVID-19 drug if proven in animal experiments and on patients.

In search of RdRp and Mpro inhibitors against SARS CoV-2: Molecular docking, molecular dynamic simulations and ADMET analysis.

Corona Virus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome coronavirus (SARS CoV-2) has been declared a worldwide pandemic by WHO recently. The complete understanding of the complex genomic structure of SARS CoV-2 has enabled the use of computational tools in search of SARS CoV-2 inhibitors against the multiple proteins responsible for its entry and multiplication in human cells. With this endeavor, 177 natural, anti-viral chemical entities and their derivatives, selected through the critical analysis of the literatures, were studied using pharmacophore screening followed by molecular docking against RNA dependent RNA polymerase and main protease. The identified hits have been subjected to molecular dynamic simulations to study the stability of ligand-protein complexes followed by ADMET analysis and Lipinski filters to confirm their drug likeliness. It has led to an important start point in the drug discovery and development of therapeutic agents against SARS CoV-2.

Needlestick-Associated Rocky Mountain Spotted Fever, Brazil.

We report a fatal case of Rocky Mountain spotted fever (RMSF) in a man in Brazil without recent history of tick bites or environmental exposure. He received an accidental needlestick while working as a nurse. The nurse and his patient died. Both cases were confirmed as RMSF by molecular methods.

Serosurvey of arthropod-borne diseases among shelter dogs in the Cumberland Gap Region of the United States.

BACKGROUND: The Cumberland Gap Region (CGR) of the United States is a natural corridor between the southeastern, northeastern, and midwestern regions of the country. CGR has also many species of ticks and mosquitos that serve as competent vectors for important animal and human pathogens. In this study, we tested dogs from six different animal shelters in the CGR for Rocky Mountain spotted fever (RMSF), anaplasmosis, Lyme disease, canine ehrlichiosis and canine heartworm disease. RESULTS: Sera from 157 shelter dogs were tested for antibodies to RMSF agent, Rickettsia rickettsii, using an indirect immunofluorescence assay. Sixty-six dogs (42.0%) were positive for either IgM or IgG, or both IgM and IgG antibodies to R. rickettsii. Moreover, the same set of sera (n = 157) plus an and additional sera (n = 75) from resident dogs at the same shelters were tested using the SNAP 4Dx Plus. Of 232 dogs tested, two (0.9%) were positive for antibodies to Anaplasma phagocytophilum/A. platys, nine (3.9%) were positive for antibodies to Borrelia burgdorferi, 23 (9.9%) for positive for antibodies to Ehrlichia canis/E. ewingii, and 13 (5.6%) were positive for Dirofilaria immitis antigen. Co-infection with two or more etiologic agents was detected in five animals. Three dogs had antibodies to both B. burgdorferi and E. canis/E. ewingii, and two dogs were positive for D. immitis antigen and antibodies to B. burgdorferi and E. canis/E. ewingii. CONCLUSIONS: Shelter dogs in the CGR are exposed to a number of important vector-borne pathogens. Further studies are required to ascertain the roles these animals play in maintenance and transmission of these pathogens.

Neuroprotective immunity by essential nutrient "Choline" for the prevention of SARS CoV2 infections: An in silico study by molecular dynamics approach.

Prenatal COVID infection is one of the worst affected and least attended aspects of the COVID-19 disease. Like other coronaviruses, CoV2 infection is anticipated to affect fetal development by maternal inflammatory response on the fetus and placenta. Studies showed that higher prenatal choline level in mother's body can safeguard the developing brain of the fetus from the adverse effects of CoV2 infection. Choline is commonly used as food supplement. By virtual screening, molecular docking and molecular dynamics techniques, we have established a strong inhibitory possibility of choline for SARS 3CLpro protease which may provide a lead for prenatal COVID-19 treatment.

Study of Caspase 8 Inhibition for the Management of Alzheimer's Disease: A Molecular Docking and Dynamics Simulation.

Alzheimer's disease (AD) is the most common type of dementia and usually manifests as diminished episodic memory and cognitive functions. Caspases are crucial mediators of neuronal death in a number of neurodegenerative diseases, and caspase 8 is considered a major therapeutic target in the context of AD. In the present study, we performed a virtual screening of 200 natural compounds by molecular docking with respect to their abilities to bind with caspase 8. Among them, rutaecarpine was found to have the highest (negative) binding energy (-6.5 kcal/mol) and was further subjected to molecular dynamics (MD) simulation analysis. Caspase 8 was determined to interact with rutaecarpine through five amino acid residues, specifically Thr337, Lys353, Val354, Phe355, and Phe356, and two hydrogen bonds (ligand: H35-A: LYS353:O and A:PHE355: N-ligand: N5). Furthermore, a 50 ns MD simulation was conducted to optimize the interaction, to predict complex flexibility, and to investigate the stability of the caspase 8-rutaecarpine complex, which appeared to be quite stable. The obtained results propose that rutaecarpine could be a lead compound that bears remarkable anti-Alzheimer's potential against caspase 8.

Rickettsia rickettsii Whole-Cell Antigens Offer Protection against Rocky Mountain Spotted Fever in the Canine Host.

Rocky Mountain spotted fever (RMSF) is a potentially fatal tick-borne disease in people and dogs. RMSF is reported in the United States and several countries in North, Central, and South America. The causative agent of this disease, Rickettsia rickettsii, is transmitted by several species of ticks, including Dermacentor andersoni, Rhipicephalus sanguineus, and Amblyomma americanum RMSF clinical signs generally include fever, headache, nausea, vomiting, muscle pain, lack of appetite, and rash. If untreated, it can quickly progress into a life-threatening illness in people and dogs, with high fatality rates ranging from 30 to 80%. While RMSF has been known for over a century, recent epidemiological data suggest that the numbers of documented cases and the fatality rates remain high in people, particularly during the last two decades in parts of North America. Currently, there are no vaccines available to prevent RMSF in either dogs or people. In this study, we investigated the efficacies of two experimental vaccines, a subunit vaccine containing two recombinant outer membrane proteins as recombinant antigens (RCA) and a whole-cell inactivated antigen vaccine (WCA), in conferring protection against virulent R. rickettsii infection challenge in a newly established canine model for RMSF. Dogs vaccinated with WCA were protected from RMSF, whereas those receiving RCA developed disease similar to that of nonvaccinated R. rickettsii-infected dogs. WCA also reduced the pathogen loads to nearly undetected levels in the blood, lungs, liver, spleen, and brain and induced bacterial antigen-specific immune responses. This study provides the first evidence of the protective ability of WCA against RMSF in dogs.

Different dynamics and pathway of disulfide bonds reduction of two human defensins, a molecular dynamics simulation study.

Human defensins are a class of antimicrobial peptides that are crucial components of the innate immune system. Both human α defensin type 5 (HD5) and human ß defensin type 3 (hBD-3) have 6 cysteine residues which form 3 pairs of disulfide bonds in oxidizing condition. Disulfide bond linking is important to the protein structure stabilization, and the disulfide bond linking and breaking order have been shown to influence protein function. In this project, microsecond long molecular dynamics simulations were performed to study the structure and dynamics of HD5 and hBD-3 wildtype and analogs which have all 3 disulfide bonds released in reducing condition. The structure of hBD-3 was found to be more dynamic and flexible than HD5, based on RMSD, RMSF, and radius of gyration calculations. The disulfide bridge breaking order of HD5 and hBD-3 in reducing condition was predicted by two kinds of methods, which gave consistent results. It was found that the disulfide bonds breaking pathways for HD5 and hBD-3 are very different. The breaking of disulfide bonds can influence the dimer interface by making the dimer structure less stable for both kinds of defensin. In order to understand the difference in dynamics and disulfide bond breaking pathway, hydrophilic and hydrophobic accessible surface areas (ASA), buried surface area between cysteine pairs, entropy of cysteine pairs, and internal energy were calculated. Comparing to the wildtype, hBD-3 analog is more hydrophobic, while HD5 is more hydrophilic. For hBD-3, the disulfide breaking is mainly entropy driven, while other factors such as the solvation effects may take the major role in controlling HD5 disulfide breaking pathway. Proteins 2017; 85:665-681. © 2016 Wiley Periodicals, Inc.

Unique Strain of Rickettsia parkeri Associated with the Hard Tick Dermacentor parumapertus Neumann in the Western United States.

In 1953, investigators at the Rocky Mountain Laboratories in Hamilton, MT, described the isolation of a spotted fever group Rickettsia (SFGR) species from Dermacentor parumapertus ticks collected from black-tailed jackrabbits (Lepus californicus) in northern Nevada. Several decades later, investigators characterized this SFGR (designated the parumapertus agent) by using mouse serotyping methods and determined that it represented a distinct rickettsial serotype closely related to Rickettsia parkeri; nonetheless, the parumapertus agent was not further characterized or studied. To our knowledge, no isolates of the parumapertus agent remain in any rickettsial culture collection, which precludes contemporary phylogenetic placement of this enigmatic SFGR. To rediscover the parumapertus agent, adult-stage D. parumapertus ticks were collected from black-tailed jackrabbits shot or encountered as roadkills in Arizona, Utah, or Texas from 2011 to 2016. A total of 339 ticks were collected and evaluated for infection with Rickettsia species. Of 112 D. parumapertus ticks collected in south Texas, 16 (14.3%) contained partial ompA sequences with the closest identity (99.6%) to Rickettsia sp. strain Atlantic rainforest Aa46, an SFGR that is closely related or identical to an SFGR species that causes a mild rickettsiosis in several states of Brazil. A pure isolate, designated strain Black Gap, was cultivated in Vero E6 cells, and sequence analysis of the rrs, gltA, sca0, sca5, and sca4 genes also revealed the closest genetic identity to Rickettsia sp. Atlantic rainforest Aa46. Phylogenetic analysis of the five concatenated rickettsial genes place Rickettsia sp. strain Black Gap and Rickettsia sp. Atlantic rainforest Aa46 with R. parkeri in a distinct and well-supported clade.IMPORTANCE We suggest that Rickettsia sp. Black Gap and Rickettsia sp. Atlantic rainforest Aa46 represent nearly identical strains of R. parkeri and that Rickettsia sp. Black Gap or a very similar strain of R. parkeri represents the parumapertus agent. The close genetic relatedness among these taxa, as well as the response of guinea pigs infected with the Black Gap strain, suggests that R. parkeri Black Gap could cause disease in humans. The identification of this organism could also account, at least in part, for the remarkable differences in severity ascribed to Rocky Mountain spotted fever (RMSF) among various regions of the American West during the early 20th century. We suggest that the wide variation in case fatality rates attributed to RMSF could have occurred by the inadvertent inclusion of cases of milder disease caused by R. parkeri Black Gap.

Protein engineering and the use of molecular modeling and simulation: the case of heterodimeric Fc engineering.

Computational and structure guided methods can make significant contributions to the development of solutions for difficult protein engineering problems, including the optimization of next generation of engineered antibodies. In this paper, we describe a contemporary industrial antibody engineering program, based on hypothesis-driven in silico protein optimization method. The foundational concepts and methods of computational protein engineering are discussed, and an example of a computational modeling and structure-guided protein engineering workflow is provided for the design of best-in-class heterodimeric Fc with high purity and favorable biophysical properties. We present the engineering rationale as well as structural and functional characterization data on these engineered designs.

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization.

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/ßig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.

Self-reported treatment practices by healthcare providers could lead to death from Rocky Mountain spotted fever.

Among 2012 Docstyle survey respondents, 80% identified doxycycline as the appropriate treatment for Rocky Mountain spotted fever in patients ≥ 8 years old, but only 35% correctly chose doxycycline in patients <8 years old. These findings raise concerns about the higher pediatric case-fatality rate of Rocky Mountain spotted fever observed nationally. Targeted education efforts are needed.