Dangerous Human Pathogen Research Articles

Genomic transfers help to decipher the ancient evolution of filoviruses and interactions with vertebrate hosts.

Although several filoviruses are dangerous human pathogens, there is conflicting evidence regarding their origins and interactions with animal hosts. Here we attempt to improve this understanding using the paleoviral record over a geological time scale, protein structure predictions, tests for evolutionary maintenance, and phylogenetic methods that alleviate sources of bias and error. We found evidence for long branch attraction bias in the L gene tree for filoviruses, and that using codon-specific models and protein structural comparisons of paleoviruses ameliorated conflict and bias. We found evidence for four ancient filoviral groups, each with extant viruses and paleoviruses with open reading frames. Furthermore, we found evidence of repeated transfers of filovirus-like elements to mouse-like rodents. A filovirus-like nucleoprotein ortholog with an open reading frame was detected in three subfamilies of spalacid rodents (present since the Miocene). We provide evidence that purifying selection is acting to maintain amino acids, protein structure and open reading frames in these elements. Our finding of extant viruses nested within phylogenetic clades of paleoviruses informs virus discovery methods and reveals the existence of Lazarus taxa among RNA viruses. Our results resolve a deep conflict in the evolutionary framework for filoviruses and reveal that genomic transfers to vertebrate hosts with potentially functional co-options have been more widespread than previously appreciated.

Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers

Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms. Apo polymerase protomers can adopt two drastically different conformations, which assemble into two distinct symmetric homodimers, that can themselves gather to form hexamers. Polymerase dimerization induces the stabilization of most polymerase domains, including the C-terminal domain that contributes the most to dimer’s interface, along with a lariat region that participates to the polymerase steadying. Binding to viral RNA induces significant conformational changes resulting in symmetric oligomer disruption and polymerase activation, suggesting the possible involvement of apo multimers as protecting systems that would stabilize the otherwise flexible C-terminal domains. Overall, these results provide insights into the multimerization capability of Hantavirus polymerase and may help to define antiviral compounds to counteract these life-threatening viruses.

Klebsiella pneumoniae DedA family proteins have redundant roles in divalent cation homeostasis and resistance to phagocytosis.

The DedA superfamily is a highly conserved family of membrane proteins. Deletion of Escherichia coli yqjA and yghB, encoding related DedA family proteins, results in sensitivity to elevated temperature, antibiotics, and alkaline pH. The human pathogen Klebsiella pneumoniae possesses genes encoding DedA family proteins with >90% amino acid identity to E. coli YqjA and YghB. We hypothesized that the deletion of K. pneumoniae yqjA and yghB will impact its physiology and may reduce its virulence. The K. pneumoniae ΔyqjA ΔyghB mutant (strain VT101) displayed a growth defect at 42°C and alkaline pH sensitivity, not unlike its E. coli counterpart. However, VT101 retained mostly wild-type resistance to antibiotics. We found VT101 was sensitive to the chelating agent EDTA, the anionic detergent SDS, and agents capable of alkalizing the bacterial cytoplasm such as bicarbonate or chloroquine. We could restore growth at alkaline pH and at elevated temperature by addition of 0.5-2 mM Ca2+ or Mg2+ to the culture media. VT101 displayed a slower uptake of calcium, which was dependent upon calcium channel activity. VT201, with similar deletions as VT101 but derived from a virulent K. pneumoniae strain, was highly susceptible to phagocytosis by alveolar macrophages and displayed a defect in the production of capsule. These findings suggest divalent cation homeostasis and virulence are interlinked by common functions of the DedA family.IMPORTANCEKlebsiella pneumoniae is a dangerous human pathogen. The DedA protein family is found in all bacteria and is a membrane transporter often required for virulence and antibiotic resistance. K. pneumoniae possesses homologs of E. coli YqjA and YghB, with 60% amino acid identity and redundant functions, which we have previously shown to be required for tolerance to biocides and alkaline pH. A K. pneumoniae strain lacking yqjA and yghB was found to be sensitive to alkaline pH, elevated temperature, and EDTA/SDS and displayed a defect in calcium uptake. Sensitivity to these conditions was reversed by addition of calcium or magnesium to the growth medium. Introduction of ΔyqjA and ΔyghB mutations into virulent K. pneumoniae resulted in the loss of capsule, increased phagocytosis by macrophages, and a partial loss of virulence. These results show that targeting the Klebsiella DedA family results in impaired divalent cation transport and, in turn, loss of virulence.

Insecticidal Property of Plumeria obtusa L. (Apocynaceae) Leaf Extracts Against Aedes aegypti (Diptera: Culicidae)

Mosquitoes are one of the most lethal animals in the world and transmit many dangerous human pathogens, causing millions of deaths each year. Aedes aegypti is generally known to be a vector of dengue, chikungunya and zika. Mosquito control is a major challenge nowadays when they became resistant against commonly available insecticides. To reduce this, it is important to look for alternative that are easily biodegradable. One such alternative could be bioactive products from phytochemicals by systematically exploring the global floral biodiversity. Therefore the present study was to evaluate the ovipositional deterrent activity of the ethanol, hexane leaf extracts of Plumeria obtusa against Aedes aegypti and also observe various types of sensilla located on the antennae, maxillary palp, tarsi and ovipositor of the Ae. aegypti using scanning electron microscope. The effect of ethanol and hexane leaf extracts of P. obtusa on oviposition of Ae. aegypti was determined under two sets of conditions (Choice and no choice oviposition assay). Among the total number of eggs laid 33.83% (ethanol), 38.15(hexane) was present in control medium when placed along with ovitraps with 0.1, 0.3, 0.7, 0.010 and 0.013% ethanol and hexane leaf extracts of P. obtusa in which appeared 27.88, 23.27, 11.79, 9.371 and 0.038 % (ethanol), 26.09, 18.14, 13.19, 4.357 and 0.042% (hexane) of eggs respectively. This was also indicated by ODI values (9.649, 18.48, 48.30, 82.86 and 99.77) (ethanol) and (18.76, 35.53, 48.59, 79.50 and 99.77) (hexane) (choice oviposition assay). Percent oviposition in 0.1, 0.3, 0.7, 0.010 and 0.013 % ethanol and hexane of leaf extracts of P. obtusa was 43.11, 28.77, 19.51, 3.875 and 0.090% (ethanol), 36.36, 26.21, 18.03, 8.888 and 0.181% (hexane) which were less compared to their control counterparts 56.88, 71.22, 80.48, 96.12 and 99.90 (ethanol), 63.63, 73.78 , 81.96 , 91.11 and 99.81 (hexane) respectively (no choice oviposition assay). The findings also revealed that both extracts (ethanol, hexane) showed promising ovipositional deterrent activity against adult Ae. aegypti.

PspA-mediated aggregation protects Streptococcus pneumoniae against desiccation on fomites.

Spn is a dangerous human pathogen capable of causing pneumonia and invasive disease. The virulence factor PspA has been studied for nearly four decades with well-established roles in pneumococcal evasion of C-reactive protein and neutralization of lactoferricin. Herein, we show that mammalian (m)GAPDH in mucosal secretions promotes aggregation of pneumococci in a PspA-dependent fashion, whereas lactoferrin counters this effect. PspA-mediated GAPDH-dependent bacterial aggregation protected Spn in nasal lavage elutes and grown in vitro from desiccation on fomites. Furthermore, surviving pneumococci within these aggregates retained their ability to colonize naïve hosts after desiccation. We report that Spn binds to and forms protein complexes on its surface composed of PspA, mGAPDH, and lactoferrin. Changes in the levels of these proteins therefore most likely have critical implications on Spn colonization, survival on fomites, and transmission.

Rapid and label-free screening of SARS-CoV-2 virus in the infected human nasopharyngeal swab samples using UiO-66-modified SPR chip

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus has become a dangerous human pathogen and caused severe illness that can lead to death. Yet, the standard practical diagnostic tools for detecting the SARS-CoV-2 virus require multi-step assay, including extraction and labeling steps. Thus, a simple and accurate detection tool is incredibly desirable to support on-site screening, preventing its spread. In this work, we developed a portable surface plasmon resonance (SPR) sensor chip modified with metal–organic frameworks (MOF) UiO-66 that serves as an immobilization matrix of single-chain variable fragment (scFv) receptors. Large surface area and good adsorption of UiO-66/scFv-modified surface lead to an improvement of binding capacity towards the protein target of the SARS-CoV-2 receptor binding domain (RBD). We further successfully demonstrated the use of this portable SPR to detect the SARS-CoV-2 virus from patients' nasopharyngeal swab samples. Interestingly, the SPR sensors can directly distinguish the positive and negative patients within 15 minutes on 23 samples, simultaneously, without the need for labeling, and the results are in line with the gold standard PCR tests. In addition, the detection limit of the sensors is 10.085 × 105 virus/mL, which follows the standard of practical screening tools. Therefore, these UiO-66 modified SPR chip sensors offer a rapid, label-free, and low-cost detection of the SARS-CoV-2 virus that can be potentially developed for other infectious diseases.

Efficacy of Acacia nilotica, Eucalyptus camaldulensis, and Salix safsafs on the mortality and development of two vector-borne mosquito species, Culex pipiens and Aedes aegypti, in the laboratory and field

Mosquitoes are one of the most lethal animals in the world and transmit many dangerous human pathogens, causing millions of deaths each year. The search for modern and better mosquito control is an endless effort almost all over the world. Phytochemicals are promising biological agents for getting rid of pests that are harmful to human and animal health and crops, they are inexpensive, biodegradable, and have diverse modes of action. The efficacy of acetone and hexane leaf extracts of Acacia nilotica, Eucalyptus camaldulensis, and Salix safsafs was investigated against the 2nd and 4th larvae and pupae of two vectors, Culex pipiens and Aedes aegypti. The results showed the obvious effect of A. nilotica extract on the mortality of mosquito larvae, the reduction of female eggs, and a higher mortality rate in sunlight than in shadow (fluorescein). Data from field trials revealed that A. nilotica extracts had the greatest effect on larval reduction, reaching 89.8% in 24 h and having a 12-day stability. Polyethylene glycol, sesquiterpenes, and fatty acids were the most common compounds found in A. nilotica, E. camaldulensis, and S. safsafs, respectively. The acacia plant had promising larvicidal activity, safe and effective alternative to chemical insecticides.

Pneumonia caused by coagulase - positive methicillin - resistant Staphylococcus aureus

Introduction. Staphylococcus (S.) aureus is one of the most omnipresent and dangerous human pathogens, whose main characteristic is the production of the enzyme coagulase. This characteristic serves to identify and assess the pathogenicity of the bacteria. In addition to skin infections, endocarditis, osteomyelitis, and infectious arthritis, it is a common cause of pneumonia both in children and adults. Case report. We described a case of a 65-year-old woman with a dry cough and malaise with patchy areas of consolidation on the chest X-ray and ?ground-glass? opacity with bronchial wall thickening and unilateral mediastinal lymphadenopathy on chest computed tomography imaging. Methicillin-resistant S. aureus was isolated from the bronchoalveolar aspirate taken during bronchoscopy. The woman was empirically treated with azithromycin, and later, based on the antibiogram findings, azithromycin was replaced with meropenem, after which her health improved. Conclusion. We presented a rare case of pneumonia with unconvincing symptomatology and laboratory and radiological findings. Paying more attention to such cases in the future is crucial, especially to the use of antibiotics to which staphylococci are increasingly developing resistance.

Crystal structure and cap binding analysis of the methyltransferase of langat virus

Tick-borne encephalitis virus (TBEV) is a major dangerous human pathogen, as TBEV infection can cause serious illness that can lead to irreversible neurological sequelae and even death. Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serogroup, belongs to the family Flaviviridae, genus Flavivirus. Its nonstructural protein 5 (NS5) protein contains a methyltransferase (MTase) domain that can methylate RNA cap structures, which is critical for viral replication. We determined the structure of LGTV NS5 methyltransferase bound to S-adenosyl-L-homocysteine (SAH) at a 1.70 Å resolution. Sequence analysis and structural comparison of homologous MTases suggests that folds and structures are closely conserved throughout Flavivirus species and play important roles. This study provides the key structural information on LGTV MTase and the foundation for research on antiviral drugs targeting LGTV MTase.

Arginine 58 is indispensable for proper function of the Francisella tularensis subsp. holarctica FSC200 HU protein, and its substitution alters virulence and mediates immunity against wild-type strain

ABSTRACT HU protein, a member of the nucleoid-associated group of proteins, is an important transcription factor in bacteria, including in the dangerous human pathogen Francisella tularensis. Generally, HU protein acts as a DNA sequence non-specific binding protein and it is responsible for winding of the DNA chain that leads to the separation of transcription units. Here, we identified potential HU protein binding sites using the ChIP-seq method and two possible binding motifs in F. tularensis subsp. holarctica FSC200 depending upon growth conditions. We also confirmed that FSC200 HU protein is able to introduce negative supercoiling of DNA in the presence of topoisomerase I. Next, we showed interaction of the HU protein with a DNA region upstream of the pigR gene and inside the clpB gene, suggesting possible regulation of PigR and ClpB expression. Moreover, we showed that arginine 58 and partially arginine 61 are important for HU protein’s DNA binding capacity, negative supercoiling induction by HU, and the length and winding of FSC200 chromosomal DNA. Finally, in order to verify biological function of the HU protein, we demonstrated that mutations in arginine 58, arginine 61, and serine 74 of the HU protein decrease virulence of FSC200 both in vitro and in vivo and that immunization using these mutant strains is able to protect as many as 100% of mice against wild-type challenge. Taken together, our findings deepen knowledge about the role of the HU protein in tularaemia pathogenesis and suggest that HU protein should be addressed in the context of tularaemia vaccine development.

Ultra-Sensitive and Rapid Detection of Pathogenic Yersinia enterocolitica Based on the CRISPR/Cas12a Nucleic Acid Identification Platform.

Yersinia enterocolitica is a dangerous foodborne human pathogen that mainly causes gastroenteritis. Ideal methods for the detection of pathogens in food should be rapid, sensitive, specific, and cost effective. To this end, novel in vitro nucleic acid identification methods based on clustered, regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) endonuclease have received increasing attention. In this study, a simple, visual, and ultrasensitive method, based on CRISPR/Cas12a with recombinase polymerase amplification (RPA), was developed for the detection of Y. enterocolitica. The results show that a specific attachment invasion locus gene (ail) can be rapidly detected using a CRISPR/Cas12a-RPA-based system. Application of the method to raw pork, which was artificially infected with Y. enterocolitica, achieved an estimated detection limit of 1.7 CFU/mL in less than 45 min, and this was 100 times lower compared with qPCR. The results indicated that the CRISPR/Cas12a-RPA system has good potential for monitoring pathogenic Y. enterocolitica in the chilled meat supply chain.

The Evolution and Biology of SARS-CoV-2 Variants.

Our understanding of the still unfolding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic would have been extremely limited without the study of the genetics and evolution of this new human coronavirus. Large-scale genome-sequencing efforts have provided close to real-time tracking of the global spread and diversification of SARS-CoV-2 since its entry into the human population in late 2019. These data have underpinned analysis of its origins, epidemiology, and adaptations to the human population: principally immune evasion and increasing transmissibility. SARS-CoV-2, despite being a new human pathogen, was highly capable of human-to-human transmission. During its rapid spread in humans, SARS-CoV-2 has evolved independent new forms, the so-called "variants of concern," that are better optimized for human-to-human transmission. The most important adaptation of the bat coronavirus progenitor of both SARS-CoV-1 and SARS-CoV-2 for human infection (and other mammals) is the use of the angiotensin-converting enzyme 2 (ACE2) receptor. Relaxed structural constraints provide plasticity to SARS-related coronavirus spike protein permitting it to accommodate significant amino acid replacements of antigenic consequence without compromising the ability to bind to ACE2. Although the bulk of research has justifiably concentrated on the viral spike protein as the main determinant of antigenic evolution and changes in transmissibility, there is accumulating evidence for the contribution of other regions of the viral proteome to virus-host interaction. Whereas levels of community transmission of recombinants compromising genetically distinct variants are at present low, when divergent variants cocirculate, recombination between SARS-CoV-2 clades is being detected, increasing the risk that viruses with new properties emerge. Applying computational and machine learning methods to genome sequence data sets to generate experimentally verifiable predictions will serve as an early warning system for novel variant surveillance and will be important in future vaccine planning. Omicron, the latest SARS-CoV-2 variant of concern, has focused attention on step change antigenic events, "shift," as opposed to incremental "drift" changes in antigenicity. Both an increase in transmissibility and antigenic shift in Omicron led to it readily causing infections in the fully vaccinated and/or previously infected. Omicron's virulence, while reduced relative to the variant of concern it replaced, Delta, is very much premised on the past immune exposure of individuals with a clear signal that boosted vaccination protects from severe disease. Currently, SARS-CoV-2 has proven itself to be a dangerous new human respiratory pathogen with an unpredictable evolutionary capacity, leading to a risk of future variants too great not to ensure all regions of the world are screened by viral genome sequencing, protected through available and affordable vaccines, and have non-punitive strategies in place for detecting and responding to novel variants of concern.

Exploring Noncovalent Protease Inhibitors for the Treatment of Severe Acute Respiratory Syndrome and Severe Acute Respiratory Syndrome-Like Coronaviruses.

Over the last 20 years, both severe acute respiratory syndrome coronavirus-1 and severe acute respiratory syndrome coronavirus-2 have transmitted from animal hosts to humans causing zoonotic outbreaks of severe disease. Both viruses originate from a group of betacoronaviruses known as subgroup 2b. The emergence of two dangerous human pathogens from this group along with previous studies illustrating the potential of other subgroup 2b members to transmit to humans has underscored the need for antiviral development against them. Coronaviruses modify the host innate immune response in part through the reversal of ubiquitination and ISGylation with their papain-like protease (PLpro). To identify unique or overarching subgroup 2b structural features or enzymatic biases, the PLpro from a subgroup 2b bat coronavirus, BtSCoV-Rf1.2004, was biochemically and structurally evaluated. This evaluation revealed that PLpros from subgroup 2b coronaviruses have narrow substrate specificity for K48 polyubiquitin and ISG15 originating from certain species. The PLpro of BtSCoV-Rf1.2004 was used as a tool alongside PLpro of CoV-1 and CoV-2 to design 30 novel noncovalent drug-like pan subgroup 2b PLpro inhibitors that included determining the effects of using previously unexplored core linkers within these compounds. Two crystal structures of BtSCoV-Rf1.2004 PLpro bound to these inhibitors aided in compound design as well as shared structural features among subgroup 2b proteases. Screening of these three subgroup 2b PLpros against this novel set of inhibitors along with cytotoxicity studies provide new directions for pan-coronavirus subgroup 2b antiviral development of PLpro inhibitors.

Chalcones from Angelica keiskei (ashitaba) inhibit key Zika virus replication proteins

Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date. The chalcones are a group of small molecules that are found in a number of different plants, including Angelica keiskei Koidzumi, also known as ashitaba. To examine chalcone anti-ZIKV activity, three chalcones, 4-hydroxyderricin (4HD), xanthoangelol (XA), and xanthoangelol-E (XA-E), were purified from a methanol-ethyl acetate extract from A. keiskei. Molecular and ensemble docking predicted that these chalcones would establish multiple interactions with residues in the catalytic and allosteric sites of ZIKV NS2B-NS3 protease, and in the allosteric site of the NS5 RNA-dependent RNA-polymerase (RdRp). Machine learning models also predicted 4HD, XA and XA-E as potential anti-ZIKV inhibitors. Enzymatic and kinetic assays confirmed chalcone inhibition of the ZIKV NS2B-NS3 protease allosteric site with IC50s from 18 to 50 µM. Activity assays also revealed that XA, but not 4HD or XA-E, inhibited the allosteric site of the RdRp, with an IC50 of 6.9 µM. Finally, we tested these chalcones for their anti-viral activity in vitro with Vero cells. 4HD and XA-E displayed anti-ZIKV activity with EC50 values of 6.6 and 22.0 µM, respectively, while XA displayed relatively weak anti-ZIKV activity with whole cells. With their simple structures and relative ease of modification, the chalcones represent attractive candidates for hit-to-lead optimization in the search of new anti-ZIKV therapeutics.

НОВИЙ БАКТЕРІОФАГ МУЛЬТИРЕЗИСТЕНТНОГО ШТАМУ PSEUDOMONAS AERUGINOSA

Aim: Antimicrobial resistance in bacteria is one of the most discussed and important theme in healthcare field now. Bacteriophages are known as the most promising alternative to antibiotics and a method of fighting the multi-resistant pathogens. In this study, our main aim was to isolate lytic specific phages of multidrug resistant Pseudomonas aeruginosa, one of the most dangerous bacterial human pathogens. Methods: Disk-diffusion method, spot-test, agar overlay method, electron microscopy. Results: Antimicrobial resistance range of P. aeruginosa 458 strain was tested and it appeared to be resistant to all available antibiotics. Phages were isolated from waste waters in Kyiv. They appeared to have a typical morphology of Myoviridae family. Isolated viruses successfully lysed all the bacteria in vitro. Conclusions: Considering high effectiveness of isolated phage in vitro, we propose it as a candidate for phage therapy, though further studies on physical and genetic characteristics are required.

Impact of the COVID-19 pandemic on mortality dynamics in the Far Eastern Federal District

Introduction. New viral respiratory infections in humans make it possible to speak of corona viruses as extremely dangerous human pathogens. They are characterized by a high mortality rate and pose a significant medical and social threat to society due to life-threatening complications of the disease. In this regard, it seems necessary to answer the questions about the dynamics of mortality of the population of the region for different classes of ICD-10 in the prepandemic and pandemic period, including various nosological forms of respiratory diseases. Aim. To assess the degree of influence of the pandemic of the new respiratory coronavirus infection COVID-19 on the mortality rate of the population for various classes of ICD-10, including the class “Diseases of the respiratory system” in the Far Eastern Federal District.Materials and methods. To implement the tasks of the study, a complex of analytical, epidemiological and statistical studies was carried out. Methods used: descriptive statistics, epidemiological analysis and monitoring; mathematical; structural and comparative analysis, methods of time series analysis and content analysis of publications on the problem. Epidemiological and statistical assessment of mortality was carried out on the basis of ICD-10 using the database of the Federal State Statistics Service for 1999-2020 in the territory of the Far Eastern Federal District. For the analysis and processing of statistical information, modern information systems and computer programs (MS Excel-2016) were used. Results. The pandemic of COVID-19 has had a significant impact on the level and structure of mortality across the entire spectrum of the main causes of death, and its magnitude (98.8 per 100,000, 2020), as a new cause of death, exceeds the whole the class of causes of death ICD-10 (J00-J99) by 33.3%, changing the pace and direction of demographic processes in Russia. At the same time, the dynamics of mortality due to respiratory diseases is unprecedented, the level of which in the Russian Federation in the first year of the COVID-19 pandemic increased by 38.8%, and in the Far Eastern Federal District – by 27.7% against the background of the previous long-term stable a downward trend in mortality rates. Especially high growth rates of mortality rates are observed in pneumonia, the level of which has increased by 3.5 times over the last decade, and among city residents – by 4.2 times (Amur Region). Features of the socio-economic and natural-climatic living conditions of the population of the region determine a significant differentiation of regions in terms of mortality rates, including due to respiratory diseases, the gradients of which among the subjects of the Far Eastern Federal District in the first year of the pandemic period (2020) amounted to 2.5 times with a minimum the level in the Republic of Sakha (Yakutia) (40.9 per 100,000 population) and the maximum – in the Jewish Autonomous Region (101.7 per 100,000 population), which indicates the ambiguous degree of influence of the new viral infection on the dynamics of mortality. At the same time, the variability in mortality rates due to respiratory diseases in the previous period (1999-2019) was even more pronounced, and among the subjects of the Far Eastern Federal District, during this period, higher mortality rates were also demonstrated, which are not even comparable to the current level of the pandemic period. These circumstances require a detailed study and a substantive analysis of the degree of influence of a COVID-19 on the mortality rate of the population using mechanisms and methods for its detection, record and registration. Conclusion. The pandemic of COVID19 had a significant impact on the level and structure of mortality across the entire spectrum of major causes of death, especially in the ICD-10 class “Respiratory diseases”, changing the pace and direction of demographic processes.

Abstract P-1: Analysis of Tick-borne Encephalitis Virus Single-particle Imaging on X-ray Free-electron Laser

Background: Tick-borne encephalitis virus (TBEV) is a dangerous human pathogen which envelope structure is already known from cryoEM study. TBEV mature viral particle size (~50 nm in diameter) makes it suitable for single-particle imaging (SPI) on X-ray free-electron laser (XFEL). XFEL SPI studies are at the early stages of development; thus, a well-described and conformationally homogeneous sample is required to develop approaches for experimental setup and data analysis. Here we present the image analysis results of data collected in October 2019 during the European XFEL experiment #2316. Methods: The detector was placed at 1.62 m from the injector; photon energy was around 6 keV, pulse energy 4 mJ, beam diameter ~ 500 nm. All runs were processed to detect hits with threshold filter (5th percentile of lit pixels) and further filtered to omit low-intensity images and images that lack detector modules. Filtered hits were background and geometry corrected with SPImage library and custom python scripts. Then hits were azimuthally integrated using PyFAI library. Scattering profiles were further clustered using the affinity propagation algorithm with cosine similarity metric in log space. Extracted classes were used to build averaged images. All hit profiles were fitted with model scattering to estimate the diameter of the particle. Simulated diffraction patterns were prepared using Condor from the cryoEM electron density map (EMDB ID 3752). Results: During the analysis after the filtering, only 276 clean and bright hits were collected per 135 min of injection (from 27287 hits detected via lit pixels threshold). Thus the hit rate was around ~ 2 hits/min, which is expected to rise in the future. The majority of hits correspond to the 40-50 nm particles (Fig. 1a), which is expected for TBEV. However, the exact size may vary due to solvent evaporation, ion condensation, and possible variability in the sample. Conclusion: The averaged images and their scattering profiles correlate with the simulated scattering patterns, though not ideally (Fig. 1 bc). Such discrepancy is expected due to the absence of electron density in the center of modeled viral structures.

Development of MEMS accelerometer based real time data acquisition prototype for COVID-19 patient breath monitoring and assessment

In late 2019, coronavirus-2, a dangerous human pathogen that causes the disease coronavirus disease 2019, arose as a severe human pathogen (COVID-19). Acute respiratory failure, which is similar to acute respiratory distress syndrome, is the most frequent clinical presentation of extreme COVID-19. COVID-19 includes diseases of the airway, lung parenchymal, pulmonary vascular, and respiratory neuromuscular systems. This essay summarises what is learned about the impact of coronavirus-2 infection on various areas of the respiratory system, provides insights into the basic pathology of respiratory disease, and highlights current and prospective translational and clinical research. This paper discusses economic and reliable respiratory monitoring systems using prototype popular embedded controller and built-in smartphone MEMS accelerometer. The hardware is tested and implemented on healthy, and Covid-19 affected persons for respiratory data, and Correlation has been made among them. One class SVM(Support Vector Machine) learning algorithm further processes the data collected from the sensor. The project primarily aims to detect anomalies in the respiratory data set to assess the rate of respiration by matching the data sets of a healthy individual and a COVID infected person.

Comprehensive comparative genomics reveals over 50 phyla of free-living and pathogenic bacteria are associated with diverse members of the amoebozoa

The Amoebozoa, a group containing predominantly amoeboid unicellular protists has been shown to play an important ecological role in controlling environmental bacteria. Amoebozoans not only graze bacteria but also serve as a safe niche for bacterial replication and harbor endosymbiotic bacteria including dangerous human pathogens. Despite their importance, only a few lineages of Amoebozoa have been studied in this regard. In this research, we conducted a comprehensive genomic and transcriptomic study with expansive taxon sampling by including representatives from the three known clades of the Amoebozoa. We used culture independent whole culture and single cell genomics/transcriptomics to investigate the association of bacteria with diverse amoebozoans. Relative to current published evidence, we recovered the largest number of bacterial phyla (64) and human pathogen genera (51) associated with the Amoebozoa. Using single cell genomics/transcriptomics we were able to determine up to 24 potential endosymbiotic bacterial phyla, some potentially endosymbionts. This includes the majority of multi-drug resistant pathogens designated as major public health threats. Our study demonstrates amoebozoans are associated with many more phylogenetically diverse bacterial phyla than previously recognized. It also shows that all amoebozoans are capable of harboring far more dangerous human pathogens than presently documented, making them of primal public health concern.

Structural Determinants of Substrate Specificity of SplF Protease from Staphylococcus aureus

Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.