Keratinase Production by Endophytic Bacteria Aneurinibacillus aneurinilyticus VRCS-4 Isolated from Xerophytic Plant Opuntia ficus - indica (Prickly pear)

Bacterial endophytes colonize an ecological niche which is unexplored site makes them suitable to produce pharmacologically active substances with vast biotechnological potential therefore, xerophytes were chosen to isolate the endophytes. In the present study forty endophytic bacterial isolates were isolated from xerophytic plants grown near poultry farms and feather dumping sites. Of them eight isolates showed zone of hydrolysis and the maximum zone of hydrolyisis of 36mm was with VRCS-4 on skimmed milk agar. This isolate exhibited efficient feather degradation and was identified as Aneurinibacillus aneurinilyticus based on its morphological, biochemical test and molecular sequencing method. The isolate was deposited in NCBI with an accession number MW227423.The isolate showed maximum en-zyme activity of 140.24U/ml at 72h, pH 7.5 and 40º C at 140 rpm. Chicken feather 1% (w/v) used as a sole source of carbon and nitrogen. Feather deg-radation by A.aneurinilyticus VRCS-4 showed 90% degradation in feather meal broth. Ours appears to be the first report on keratinase production by endophytic bacteria from xerophytic plant (Opuntia ficus -indica).

Scenes From Tick Physiology: Proteins of Sialome Talk About Their Biological Processes

Ticks are blood-sucking parasites with different strategies of feeding depending on the tick family. The major families are Ixodidae or Argasidae, being slow or fast feeders, respectively. In the recent years, the advances in molecular sequencing techniques have enabled to gain knowledge about the proteome of the tick’s salivary glands. But an holistic view of the biological processes underlying the expression of the sialome has been neglected. In this study we propose the use of standard biological processes as a tool to draw the physiology of the tick’s salivary glands. We used published data on the sialome of Rhipicephalus sanguineus s.l. (Ixodidae) and Ornithodoros rostratus (Argasidae). A partial set of proteins obtained by these studies were used to define the biological process(es) in which proteins are involved. We used a directed network construction in which the nodes are proteins (source) and biological processes (target), separately for the low-level processes (“children”) and the top-level ones (“parents”). We applied the method to feeding R. sanguineus at different time slices, and to different organs of O. rostratus. The network connects the proteins and the processes with a strength directly proportional to the transcript per millions of each protein. We used PageRank as a measure of the importance of each biological process. As suggested in previous studies, the sialome of unfed R. sanguineus express about 30% less biological processes than feeding ticks. Another decrease (25%) is noticed at the middle of the feeding and before detachment. However, top-level processes are deeply affected only at the onset of feeding, demonstrating a redundancy in the feeding. When ixodid-argasid are compared, large differences were observed: they do not share 91% of proteins, but share 90% of the biological processes. However, caution must be observed when examining these results. The hypothesis of different proteins linked to similar biological process(es) in both ticks is an extreme not confirmed in this study. Considering the limitations of this study, carried out with a selected set of proteins, we propose the networks of proteins of sialome linked to their biological processes as a tool aimed to explain the biological processes behind families of proteins.

Comprehensive Investigation of a Toxic Microcystis sp. (Cyanobacteria) Collected from Snow Lake (New Mexico, USA).

The toxin producing cyanobacterium Microcystis sp. was collected in the mid October 2020 from the shallow waters of Snow Lake (New Mexico, USA). This species caused a visible bloom consisting of the pale green irregular macro colonies. Mass spectral analysis of the biomass revealed the presence of 4 derivatives of microcystin in that bloom: MC-LR (in the water and biomass), MC-RR (in biomass), MC-LY (in biomass), and MC-YR (in biomass).Next-generation sequencing allowed the retrieval of two Microcystis sequences in the bloom; which are molecular benchmarks for toxic Microcystis that may be used in future monitoring studies. Light microscopy provided evidence for the taxonomic affiliation of the found morphotype as Microcystis flos-aquae (Wittrock) Kirchner. However, molecular sequencing and the present situation in cyanobacterial taxonomy prevented affiliation of our morphotype to Microcystis flos-aquae, justifying following name – Microcystis sp. Confocal microscopy was used to determine the distribution of the cell content utilizing 3D stereo imaging. Emission spectra analysis identified the pigment composition and pigment distribution within the cells. SEM revealed 3D arrangement of the cells in the colonies, texture of the surface of the cells (perhaps dehydrated collapsed polysaccharides), F-layer and pili-like structures. Additionally, SEM/EDS analysis confirmed the F-layer using elemental composition analysis, which showed sulfur in the F-layer – typical element for that structure. Through the use of AFM, we analyzed the texture of the cell's surface and confirmed pili-like structures.

High Grade Meningiomas: Current Therapy Based on Tumor Biology

Atypical (WHO grade II) and malignant meningiomas (WHO Grade III) are a rare subset of primary intracranial tumors. Due to the high recurrence rate after surgical resection and radiotherapy, there has been a recent interest in exploring other systemic treatment options for these refractory tumors. Recent advances in molecular sequencing of tumors have elucidated new pathways and drug targets currently being studied. This article provides a thorough overview of novel investigational therapeutics, including targeted therapy, immunotherapy, and new technological modalities for atypical and malignant meningiomas. There is encouraging preclinical evidence regarding the efficacy of the emerging treatments discussed in this chapter. Several clinical trials are currently recruiting patients to translate targeted molecular therapy for recurrent and high-grade meningiomas.

Rhyacophila siparantum sp. nov. (Trichoptera: Rhyacophilidae), a new species of the R. philopotamoides species group from the Republic of Kosovo with molecular and ecological notes

In this study we describe Rhyacophila siparantum sp. nov., a new species of the Rhyacophila philopotamoides species group from the Bjeshkët e Nemuna Mountains in Kosovo, based on morphological (male adults) and molecular (sequencing of the barcode region of the cytochrome c oxidase subunit I gene (COI)) characteristics. The new species is morphologically closest to Rhyacophila schmidinarica Urbanič, Krušnik & Malicky, 2000, and also closely resembling Rhyacophila hirticornis McLachlan, 1879. R. siparantum sp. nov. differs from both its most similar congeners primarily by the shape of the segment X, which is in lateral view short apically and with a pronounced lateral hump on the posterior edge. This difference, combined with other characters related to the shape of the second segment of inferior appendages, spine pattern of parameres and size of ventral teeth on segments VI and VII, make R. siparantum sp. nov. easily distinguishable from both most similar congeners. Phylogenetic and taxonomic relationships were reconstructed using two methods of phylogenetic inference, and two species delimitation methods. All this supports Rhyacophila siparantum sp. nov. as a distinct taxon. The adults of Rhyacophila siparantum sp. nov. were found during the period May – August, nearby a small rheocrene spring inside a forested area. The new species is most probably a microendemic of the Bjeshkët e Nemuna, a mountainous massive known for several other endemic species of caddisflies. Rhyacophila siparantum sp. nov. is the seventeenth known species of the genus Rhyacophila Pictet, 1834 from Kosovo.

Evidence-Based Minireview: Molecular precision and clinical uncertainty: should molecular profiling be routinely used to guide risk stratification in MDS?

This is a focused clinical vignette and review of the literature in MDS to discuss the application of molecular sequencing for risk stratification in MDS. The authors utilize an exemplar patient case and explain the advantages and disadvantages, based on available data, of routine use of this testing for MDS patients.

Molecular-based precision oncology clinical decision making augmented by artificial intelligence

The rapid growth and decreasing cost of Next-generation sequencing (NGS) technologies have made it possible to conduct routine large panel genomic sequencing in many disease settings, especially in the oncology domain. Furthermore, it is now known that optimal disease management of patients depends on individualized cancer treatment guided by comprehensive molecular testing. However, translating results from molecular sequencing reports into actionable clinical insights remains a challenge to most clinicians. In this review, we discuss about some representative systems that leverage artificial intelligence (AI) to facilitate some processes of clinicians’ decision making based upon molecular data, focusing on their application in precision oncology. Some limitations and pitfalls of the current application of AI in clinical decision making are also discussed.

Clean room microbiome complexity impacts planetary protection bioburden

Background The Spacecraft Assembly Facility (SAF) at the NASA’s Jet Propulsion Laboratory is the primary cleanroom facility used in the construction of some of the planetary protection (PP)-sensitive missions developed by NASA, including the Mars 2020 Perseverance Rover that launched in July 2020. SAF floor samples (n=98) were collected, over a 6-month period in 2016 prior to the construction of the Mars rover subsystems, to better understand the temporal and spatial distribution of bacterial populations (total, viable, cultivable, and spore) in this unique cleanroom. Results Cleanroom samples were examined for total (living and dead) and viable (living only) microbial populations using molecular approaches and cultured isolates employing the traditional NASA standard spore assay (NSA), which predominantly isolated spores. The 130 NSA isolates were represented by 16 bacterial genera, of which 97% were identified as spore-formers via Sanger sequencing. The most spatially abundant isolate was Bacillus subtilis, and the most temporally abundant spore-former was Virgibacillus panthothenticus. The 16S rRNA gene-targeted amplicon sequencing detected 51 additional genera not found in the NSA method. The amplicon sequencing of the samples treated with propidium monoazide (PMA), which would differentiate between viable and dead organisms, revealed a total of 54 genera: 46 viable non-spore forming genera and 8 viable spore forming genera in these samples. The microbial diversity generated by the amplicon sequencing corresponded to ~86% non-spore-formers and ~14% spore-formers. The most common spatially distributed genera were Sphinigobium, Geobacillus, and Bacillus whereas temporally distributed common genera were Acinetobacter, Geobacilllus, and Bacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent being Acinetobacter. Conclusion This study clearly established that detecting spores via NSA does not provide a complete assessment for the cleanliness of spacecraft-associated environments since it failed to detect several PP-relevant genera that were only recovered via molecular methods. This highlights the importance of a methodological paradigm shift to appropriately monitor bioburden in cleanrooms for not only the aeronautical industry but also for pharmaceutical, medical industries, etc., and the need to employ molecular sequencing to complement traditional culture-based assays.

A checklist of halacarid and hydrachnid mites (Acari, Halacaridae & Hydrachnidia) associated with sponges (Porifera)

A compilation of halacarid and hydrachnid mite species found associated with sponges has been carried out based on published records. Altogether 78 halacarid mites species belonging to 15 genera viz. Agaue (9 species), Agauopsis (7 species), Arhodeoporus (1 species), Atelopsalis (1 species), Bradyagaue (1 species), Copidognathus (27 species), Halacarellus (7 species), Halacaropsis (2 species), Halacarus (5 species), Lohmannella (3 species), Maracarus (3 species), Rhombognathides (3 species), Rhombognathus (4 species), Spongihalacarus (1 species), and Thalassarachna (4 species) are reported. Twenty-four hydrachnid mite species belonging to three families viz. Hygrobatidae (2 species), Unionicolidae (20 species) and Pontarachnidae (2 species) are included. Some species of Unionicolidae are mentioned as possible sponge-mite species. Further studies, emphasizing developmental studies of unionicolid mites to get better ideas about associations with freshwater sponges are needed. Molecular sequencing will reveal more cryptic species and improve the quality of re-descriptions of currently recognized species in these sponge associated mites.

1018. Bacterial Bioburden Characterization of Infected Diabetic Foot Ulcers in Hospitalized Patients in Association with Clinical Outcomes: Traditional Cultures vs. Molecular Sequencing Methods

Background Infected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus. These potentially limb-threatening ulcers are challenging to treat due to the impairment of wound healing in diabetic patients and the complex microbial environment characterizing these ulcers. Our aim was to analyze the microbiome of IDFU in association with clinical outcomes. Methods Wound biopsies from IDFU were obtained from hospitalized patients and were analyzed using traditional microbiology cultures, 16S rRNA sequencing and shotgun metagenomic sequencing. Patients’ characteristics, culture-based results and sequencing data were analyzed in association with clinical outcomes. Study Design Results 31 patients were enrolled. Significantly more anaerobic and Gram-negative bacteria were detected with sequencing methods compared to conventional cultures (59% and 76% were anaerobes according to 16SrRNA and metagenomic respectively vs. 26% in cultures, p=0.001, and 79%, 59% and 54% were Gram negative bacteria respectively, p< 0.001). Culture-based results showed that Staphylococcus aureus was more prevalent among patients who were conservatively treated (p=0.048). In metagenomic analysis the Bacteroides genus was more prevalent among patients who underwent toe amputation (p< 0.001). Analysis of metagenomic-based functional data showed that antibiotic resistance genes and genes related to biofilm production and to bacterial virulent factors were more prevalent in IDFU that resulted in toe amputation (p< 0.001). Occurrences and mean relative abundances of the most prevalent bacteria of IDFU Comparison between [A] traditional cultures, 16S rRNA sequencing and metagenomic sequencing results (genera level - 12 samples) [B] traditional cultures and metagenomic sequencing results (species level – 30 samples) [C] traditional cultures and 16S rRNA sequencing results (genera level - 30 samples) CUL – cultures; 16S - 16S rRNA sequencing; MTG – metagenomic sequencing Bacteroides genus association with toe amputation Bacteroides genera was more common among samples of patients who underwent toe amputation compared with samples of patients who were conservatively treated (p < 0.001). Species level analysis showed that Bacteroides fragilis and Bacteroides xylanisolvens predominated IDFU of patients who underwent toe amputation (p=0.04, p=0.002 respectively). No – conservative treatment; Yes – toe amputation. Functional genes differentiating patients who underwent toe amputation from conservatively treated Yellow stars – indicate genes that were associated with bacterial virulent factors, biofilm formation and resistant mechanisms – all were more prevalent in patients who underwent toe amputation (with p values<> Conclusion Molecular sequencing tools uncover the complex biodiversity of IDFU and emphasize the high prevalence of anaerobes and Gram-negative bacteria in these ulcers. Furthermore, sequencing results highlighted the possible association between certain genera, species, and bacterial functional genes to clinical outcomes Disclosures Yossi Paitan, PhD, Ilex Medical Ltd (Employee, Other Financial or Material Support, As of 01.01.2021 I am the Laboratories Manager of Ilex Labs)