Description of B abesia coryicola sp. nov. from Florida pumas (Puma concolor coryi) from southern Florida, USA.

Previously, a high prevalence of piroplasms has been reported from Florida pumas (Puma concolor coryi) from southern Florida. In the current study, we describe the biological characteristics of a novel Babesia species in Florida pumas. Ring-stage trophozoites were morphologically similar to trophozoites of numerous small babesids of felids including B. leo, B. felis, and Cytauxzoon felis. Parasitemias in Florida pumas were very low (<1%) and hematologic values of 25 Babesia-infected Florida pumas were within normal ranges for P. concolor. Phylogenetic analysis of near full-length 18S rRNA gene, ß-tubulin, cytochrome c oxidase subunit I, cytochrome c oxidase subunit III, and cytochrome b gene sequences indicated that this Babesia species is a member of the Babesia sensu stricto clade and is related to groups of Babesia spp. from carnivores or ungulates, although the closest group varied by gene target. Internal transcribed spacer (ITS)-1 region sequences from this Babesia sp. from 19 Florida pumas were 85.7-99.5% similar to each other and ∼88% similar to B. odocoilei. Similarly, an ITS-2 sequence from one puma was 96% similar to B. bigemina and 92% similar to a Babesia sp. from a red panda (Ailurus fulgens). Infected pumas were positive for antibodies that reacted with B. odocoilei, B. canis, and B. bovis antigens with titers of 1:256, 1:128, and 1:128, respectively. No serologic reactivity was noted for Theileria equi. No molecular evidence of congenital infection was detected in 24 kittens born to 11 Babesia-infected female pumas. Pumas from other populations in the United States [Louisiana (n = 1), North Dakota (n = 5) and Texas (n = 28)], British Columbia, Canada (n = 9), and Costa Rica (n = 2) were negative for this Babesia sp. Collectively, these data provide morphologic, serologic, genetic, and natural history data for this novel Babesia sp. which we propose the name Babesia coryicola sp. nov. sp. This is the first description of a felid-associated Babesia species in North America.

Zebrafish Thrombocyte Transcriptome Analysis and Functional Genomics.

Our laboratory is interested in investigating the maturation process of zebrafish thrombocytes, which are functional equivalents to human platelets. We have adopted the zebrafish model to gain insights into mammalian platelet production, or thrombopoiesis. Notably, zebrafish exhibit two distinct populations of thrombocytes in their circulating blood: young and mature thrombocytes. This observation is intriguing because maturation appears to occur in circulation, yet the precise mechanisms governing this maturation remain elusive. Our goal is to understand the mechanisms underlying thrombocyte maturation by conducting single-cell RNA sequencing (scRNA-Seq) on young and mature thrombocytes, analyzing these transcriptomes to identify genes specific to each thrombocyte population, and elucidating the role of these genes in the maturation process, by quantifying thrombocyte numbers after the piggyback knockdown of each of these genes. In this chapter, we present a comprehensive, step-by-step protocol detailing the multifaceted methodology involved in understanding thrombocyte maturation, which encompasses the collection of zebrafish blood, the separation of young and mature thrombocytes using flow cytometry, scRNA-Seq analysis of these distinct thrombocyte populations, identification of genes specific to young and mature thrombocytes, and subsequent validation through gene knockdown techniques.

Multi-generational benefits of genetic rescue.

Genetic rescue-an increase in population fitness following the introduction of new alleles-has been proven to ameliorate inbreeding depression in small, isolated populations, yet is rarely applied as a conservation tool. A lingering question regarding genetic rescue in wildlife conservation is how long beneficial effects persist in admixed populations. Using data collected over 40 years from 1192 endangered Florida panthers (Puma concolor coryi) across nine generations, we show that the experimental genetic rescue implemented in 1995-via the release of eight female pumas from Texas-alleviated morphological, genetic, and demographic correlates of inbreeding depression, subsequently preventing extirpation of the population. We present unequivocal evidence, for the first time in any terrestrial vertebrate, that genetic and phenotypic benefits of genetic rescue remain in this population after five generations of admixture, which helped increase panther abundance (> fivefold) and genetic effective population size (> 20-fold). Additionally, even with extensive admixture, microsatellite allele frequencies in the population continue to support the distinctness of Florida panthers from other North American puma populations, including Texas. Although threats including habitat loss, human-wildlife conflict, and infectious diseases are challenges to many imperiled populations, our results suggest genetic rescue can serve as an effective, multi-generational tool for conservation of small, isolated populations facing extinction from inbreeding.

Torquetenovirus Viremia Quantification Using Real-Time PCR Developed on a Fully Automated, Random-Access Platform.

Quantification of Torquetenovirus (TTV) viremia is becoming important for evaluating the status of the immune system in solid organ transplant recipients, monitoring the appearance of post-transplant complications, and controlling the efficacy of maintenance immunosuppressive therapy. Thus, diagnostic approaches able to scale up TTV quantification are needed. Here, we report on the development and validation of a real-time PCR assay for TTV quantification on the Hologic Panther Fusion® System by utilizing its open-access channel. The manual real-time PCR previously developed in our laboratories was optimized to detect TTV DNA on the Hologic Panther Fusion® System. The assay was validated using clinical samples. The automated TTV assay has a limit of detection of 1.6 log copies per ml of serum. Using 112 samples previously tested via manual real-time PCR, the concordance in TTV detection was 93% between the assays. When the TTV levels were compared, the overall agreement between the methods, as assessed using Passing-Bablok linear regression and Bland-Altman analyses, was excellent. In summary, we validated a highly sensitive and accurate method for the diagnostic use of TTV quantification on a fully automated Hologic Panther Fusion® System. This will greatly improve the turnaround time for TTV testing and better support the laboratory diagnosis of this new viral biomarker.

Prevalence of genital high-risk human papillomavirus infections and associated factors among women living with human immunodeficiency virus in Uganda.

BACKGROUND: Women living with HIV are at risk for cervical dysplasia and cancer worldwide. In 2015, the World Health Organization (WHO) recommended that testing for high-risk HPV (hrHPV) infection be incorporated into cervical cancer screening programs using molecular nucleic acid tests (NATs) but this has not previously been done in Uganda. The country's coverage for Human Papilloma Virus (HPV) screening remains low at less than 10% for women aged 25-49 years. This study determined the genital prevalence of hrHPV infection and the associated factors among women living with HIV in Uganda. METHODS: A descriptive cross-sectional study was conducted in 15 selected health facilities among participants who were on Antiretroviral therapy (ART). Participants who consented to participate were instructed on how to collect their own high vaginal swabs using a cervical brush for HPV molecular testing (HPV DNA or HPV RNA) and their demographics data was collected using a standard questionnaire. Laboratory diagnosis for HPV molecular testing was done using Gene xpert machines and Hologic Aptima Machine. Modified Poisson regression analysis was conducted to determine the associated factors. RESULTS: This study involved 5856 HIV positive participants on ART. A total of 2006 out of 5856 (34.3%) participants had high risk HPV infections. HPV infections by genotypes were: HPV16 317(15.8%), HPV 18/45 308 (15.4%) and other high-risk HPV 1381 (68.8%). The independent factors associated with all hrHPV were parity, education level, having more than one partner, and engaging in early sex. Smoking was associated with HPV 16, HPV 18/45 and other hrHPV. Age was associated with all hrHPV, marital status with HPV 16, and occupation with HPV 16. CONCLUSIONS: The prevalence of genital high-risk HPV infections among HIV positive women attending ART clinics in public facilities in Uganda was high. Other hrHPV genotype was the commonest compared to 18/45 and HPV 16. The integration of cervical cancer screening in ART programmes remains paramount to support the early detection of cervical cancer and Non-invasive self-collected urine and vaginal sampling for cervical cancer screening present an opportunity.

Proteomic-Based Studies on Memory Formation in Normal and Neurodegenerative Disease-Affected Brains.

A critical aspect of cognition is the ability to acquire, consolidate, and evoke memories, which is considerably impaired by neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. These mnemonic processes are dependent on signaling cascades, which involve protein expression and degradation. Recent mass spectrometry (MS)-based proteomics has opened a range of possibilities for the study of memory formation and impairment, making it possible to research protein systems not studied before. However, in the context of synaptic proteome related to learning processes and memory formation, a deeper understanding of the synaptic proteome temporal dynamics after induction of synaptic plasticity and the molecular changes underlying the cognitive deficits seen in neurodegenerative diseases is needed. This review analyzes the applications of proteomics for understanding memory processes in both normal and neurodegenerative conditions. Moreover, the most critical experimental studies have been summarized using the PANTHER overrepresentation test. Finally, limitations associated with investigations of memory studies in physiological and neurodegenerative disorders have also been discussed.

Photographic documentation of melanism in bobcats (Lynx rufus) in the Greater Everglades.

We document the presence of bobcats (Lynx rufus) that demonstrate melanism in the Greater Everglades. The South Florida landscape is driven by a myriad of disturbance regimes particularly that of short fire intervals. We monitored 180 camera traps for 3 years and obtained 9503 photographs of bobcats 25 (<0.5%) of these detections included melanistic individuals. Our observations and historical accounts suggest melanism is a phenotype that persists, albeit it at an exceedingly low frequency, in bobcats in the region. While we do not know if the expression of melanism conferred a fitness benefit in our system, the vegetation structure that was characterized by frequently burned uplands and low-light and densely vegetated swamps produced conditions that may render a benefit from melanism through enhanced crypsis. The investigation of rare phenomenon in ecology is important yet difficult within a given field study, but reporting novel observations, like melanism in bobcats, allows for science to gain insight across studies that would not be otherwise possible.

A Laboratory-Developed Assay for Clade II Human Mpox Virus on the Panther Fusion Open Access System.

This paper describes the methods for developing and optimizing a laboratory-developed assay (LDA) for detecting clade II human mpox virus using the automated Panther Fusion platform and Open Access software. Various concentrations of reagents in a primer-probe mix were tested to optimize the LDA. The LDA was validated using 10 previously characterized positive and 10 negative human mpox samples, resulting in 95% accuracy and 100% precision. The LDA resulted in 100% specificity among previously tested HSV1-, HSV2-, and VZV-positive human samples. Several spiked media extensions were also validated and achieved 98% accuracy and 100% precision across all collection media types. The assay's limit of detection was calculated to be 1.475 copies/reaction, and the polymerase chain reaction efficiency resulted in 89.87% (slope, -3.5911; R2 = 0.9947). The methods described here can be applied to the rapid optimization and development of LDAs for many possible pathogens of public health importance.

Multi-center clinical evaluation of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab specimens from symptomatic individuals.

The symptomology is overlapping for respiratory infections due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A/B viruses, and respiratory syncytial virus (RSV). Accurate detection is essential for proper medical management decisions. This study evaluated the clinical performance of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab (NPS) specimens from individuals of all ages with signs and symptoms of respiratory infection consistent with COVID-19, influenza, or RSV. Retrospective known-positive and prospectively obtained residual NPS specimens were collected during two respiratory seasons in the USA. Clinical performance was established by comparing Panther Fusion SARS-CoV-2/Flu assay results to a three-molecular assay composite comparator interpretation for SARS-CoV-2 and to the FDA-cleared Panther Fusion Flu A/B/RSV assay results for all non-SARS-CoV-2 targets. A total of 1,900 prospective and 95 retrospective NPS specimens were included in the analyses. The overall prevalence in prospectively obtained specimens was 20.7% for SARS-CoV-2, 6.7% for influenza A, and 0.7% for RSV; all influenza B-positive specimens were retrospective specimens. The positive percent agreement of the Panther Fusion assay was 96.9% (378/390) for SARS-CoV-2, 98.0% (121/123) for influenza A virus, 95.2% (20/21) for influenza B virus, and 96.6% (57/59) for RSV. The negative percent agreement was ≥98.5% for all target viruses. Specimens with discordant Panther Fusion SARS/Flu/RSV assay results all had cycle threshold values of ≥32.4 (by comparator or by Panther Fusion SARS/Flu/RSV assay). Only five co-infections were detected in the study specimens. The Panther Fusion SARS-CoV-2/Flu/RSV assay provides highly sensitive and specific detection of SARS-CoV-2, influenza A virus, influenza B virus, and RSV in NPS specimens.

Role of Hologic® Panther AptimaTM SARS-CoV-2 assay in the detection of SARS-CoV-2: screening or diagnostic technique?

During the multiple waves of COVID-19 suffered all over the world, having a rapid and sensitive diagnostic test has become a priority for microbiology laboratories. The AptimaTM SARS-CoV-2 transcription-mediated amplification (TMA) assay running on the Panther system (Hologic) was presented as a very good option to cover this need. To evaluate this system, 570 respiratory samples were included in the study and were processed both by the Panther (Hologic) system and by qRT-PCR (Thermo Fisher Science, Waltham, USA), current assay for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A high number of false positives (n=76) was obtained with Panther system (Hologic), but the number of false positives decreases as the relative light units (RLU) value increases. These results show that this technique can be a good option for sample screening but checking for positive results should be mandatory, especially those with low RLU values.

Verification of dried blood spot as a sample type for HIV viral load and early infant diagnosis on Hologic Panther in Zambia.

OBJECTIVE: Zambia has embarked on improving the diagnostic capacity by setting up high throughput and accurate machines in the testing process and introduction of dried blood spot (DBS) as a sample type. This was a cross sectional study to verify dried blood spot as a sample type for HIV viral load and early infant diagnosis (EID) on Hologic Panther platform and Evaluate the analytical performance (precision, linearity and measurement of uncertainty) of the Hologic Panther. RESULTS: The specificity and sensitivity of EID performance of Aptima Quant Dx assay on Hologic panther machine against the gold standard machine COBAS Taqman (CAP/CTM) was 100% with an overall agreement of 100%. The quantitative HIV Viral Load (VL) accuracy had a positive correlation of (0.96) obtained against the gold standard (plasma samples) run on COBAS4800 platform. Analytical performance of the Hologic panther machine was evaluated; Precision low positive repeatability 3.50154 and within lab 2.268915 at mean 2.88 concentration and precision high positive repeatability 1.116955 and within lab 2.010677 at mean 5.09 concentration were obtained confirming manufacturers claims. Uncertainty of measurement for this study was found to be ± 71 copies/ml. Linearity studies were determined and all points were within acceptable limits. We therefore recommend DBS as a sample type alternative to plasma for the estimation of HIV-1 viral load and EID diagnosis on the Hologic panther machine.

Performance evaluation of the Aptima CMV quant assay using plasma and non-plasma samples.

BACKGROUND: Cytomegalovirus (CMV) infection has a major negative impact on transplantation and is associated with increased morbidity and mortality in this patient population. Quantitation of CMV infections using a molecular test is the preferred method for monitoring patients post-transplant. For this analysis, we compared the Aptima CMV Quant Assay (Aptima CMV) on the Panther system to the ELITech MGB Alert® CMV 3.0 ASR (MGB CMV) run on the ELITe InGenius®. METHODS: The analytical performance of the assay was assessed using commercially available CMV reference panels that meet the 1st WHO International Standard for Human Cytomegalovirus for nucleic acid amplification techniques. The clinical performance of the assay was determined using 249 plasma and non-plasma samples. RESULTS: The 95% LOD of the Aptima assay was determined to be 50 IU/mL and 200 IU/mL for the MGB CMV assay. A strong linear correlation with the reference panel (R2 = 0.9945), excellent reproducibility, and accuracy (R2 = 0.986) over the detection range of the assay was observed. Of the 249 clinical samples tested, only 17 (6.8%) yielded discordant results which were at or near the lower limit of quantification of the assays. Although the Aptima CMV assay demonstrated excellent concordance of qualitative results to the MGB CMV assay for all samples, the MGB CMV quantified CMV DNA at an average of 0.5 Log IU/mL higher than Aptima CMV. CONCLUSION: The Aptima CMV assay is both sensitive and accurate in quantifying CMV in both plasma and non-plasma specimens on the fully automated Panther system.

Development of real-time RT-PCR assays to detect measles virus on the Hologic Panther Fusion® System.

BACKGROUND: In 2019, Aotearoa New Zealand (NZ) experienced its worst measles outbreak since 1997. Due to declining childhood vaccination rates since the beginning of the SARS-CoV-2 pandemic, NZ is at serious risk of another major measles outbreak. Our laboratory provides diagnostic services to NZ's Southern region. In 2019 the Southern region experienced the greatest number of cases outside of Auckland and Northland, however we did not have a validated measles PCR assay in our laboratory. OBJECTIVES: We sought to develop reverse transcription real-time polymerase chain reaction (RT-PCR) assays for measles on the Hologic Panther Fusion® System by utilising its open access function. STUDY DESIGN: Previously published real-time RT-PCR assays were modified and optimised to detect wild-type measles virus (LDT-Mea), and the vaccine strain of measles virus (LDT-MeaVacA), on the Hologic Panther Fusion® System. The assays were clinically validated. RESULTS: The LDT-Mea assay has a limit of detection (LoD) of 0.1 CCID50, while the LDT-MeaVacA assay is less sensitive with a LoD of 1 CCID50. Using 27 samples, the clinical sensitivity and specificity was 100% for both assays. Other common respiratory viruses were found not to cross-react with either the LDT-Mea or LDT-MeaVacA assays. CONCLUSION: We have successfully adapted and validated for diagnostic use on the Hologic Panther Fusion® System previously published assays to detect wild-type and vaccine strains of the measles virus. The implementation of measles testing on this system will greatly improve the turn-around time for measles testing, and better support the measles public health response, for our region.

Hippocampal RNA sequencing in mice selectively bred for high and low activity.

High and Low Activity strains of mice were bidirectionally selected for differences in open-field activity (DeFries et al., 1978, Behavior Genetics, 8: 3-13) and subsequently inbred to use as a genetic model for studying anxiety-like behaviors (Booher et al., 2021, Genes, Brain and Behavior, 20: e12730). Hippocampal RNA-sequencing of the High and Low Activity mice identified 3901 differentially expressed protein-coding genes, with both sex-dependent and sex-independent effects. Functional enrichment analysis (PANTHER) highlighted 15 gene ontology terms, which allowed us to create a narrow list of 264 top candidate genes. Of the top candidate genes, 46 encoded four Complexes (I, II, IV and V) and two electron carriers (cytochrome c and ubiquinone) of the mitochondrial oxidative phosphorylation process. The most striking results were in the female high anxiety, Low Activity mice, where 39/46 genes relating to oxidative phosphorylation were upregulated. In addition, comparison of our top candidate genes with two previously curated High and Low Activity gene lists highlight 24 overlapping genes, where Ndufa13, which encodes the supernumerary subunit A13 of complex I, was the only gene to be included in all three lists. Mitochondrial dysfunction has recently been implicated as both a cause and effect of anxiety-related disorders and thus should be further explored as a possible novel pharmaceutical treatment for anxiety disorders.

Automated Real-Time PCR Detection of Tickborne Diseases Using the Panther Fusion Open Access System.

The incidence of tickborne infections in the United States has risen significantly. Automation is needed for the increasing demand for testing. The Panther Fusion (Fusion) has an Open Access functionality to perform lab developed tests (LDTs) on a fully automated system. Our laboratory adapted two LDTs on Fusion; a multiplex real-time PCR for Anaplasma phagocytophilum and Ehrlichia chaffeensis (AP/EC) and a Babesia microti (BM) PCR. Limits of detection (LODs) were performed with target region plasmid panels spiked into whole blood. The LODs for AP, BM, and EC on the Fusion were 11, 17, and 10 copies/reaction, respectively. The performance of AP/EC was evaluated with 80 whole blood specimens, including 50 specimens previously positive for AP by our test of record (TOR) and 30 specimens (including 20 AP positive) spiked with EC plasmid. AP was detected in 49 out of 50 positive specimens and EC was detected in all 30 spiked specimens. BM PCR on Fusion was evaluated with 75 whole blood samples, including 16 specimens previously shown to be positive for BM and 59 negative specimens, of which 29 were spiked with BM plasmid DNA. BM was detected in 45 samples as expected. AP/EC and BM PCRs were successfully developed and optimized on the Panther Fusion with performance characteristics comparable to our TOR. These assays complement each other and allow for a modular testing approach for tickborne diseases which have differing clinical presentation. Furthermore, automation of these assays will help the lab meet the increasing demand for testing. IMPORTANCE Since the incidence of tickborne diseases has been accelerating in the United States, automation for testing has become essential in affected regions. Unfortunately, because the need is regional, commercial test manufacturers have not yet provided answers for clinical laboratories. Here, we describe the development of PCR tests on the highly automated Panther Fusion for three tickborne diseases. The Panther Fusion assays were evaluated using 155 archived whole blood (WB) specimens previously tested for Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Babesia microti, while WB spiked with DNA from plasmid clones of the target regions were used for analytical sensitivity. We demonstrated that the Panther Fusion assays performed similar to the manual PCR tests used clinically in our laboratory and that automation of these tests had no adverse effect on the performance.

Feline Leukemia Virus Frequently Spills Over from Domestic Cats to North American Pumas.

Feline leukemia virus (FeLV) is a gammaretrovirus with horizontally transmitted and endogenous forms. Domestic cats are the primary reservoir species, but FeLV outbreaks in endangered Florida panthers and Iberian lynxes have resulted in mortalities. To assess prevalence and interspecific/intraspecific transmission, we conducted an extensive survey and phylogenetic analysis of FeLV infection in free-ranging pumas (n = 641) and bobcats (n = 212) and shelter domestic cats (n = 304). Samples were collected from coincident habitats across the United States between 1985 and 2018. FeLV infection was detected in 3.12% of the puma samples, 0.47% of the bobcat samples, and 6.25% of the domestic cat samples analyzed. Puma prevalence varied by location, with Florida having the highest rate of infection. FeLV env sequences revealed variation among isolates, and we identified two distinct clades. Both progressive and regressive infections were identified in cats and pumas. Based on the time and location of sampling and phylogenetic analysis, we inferred 3 spillover events between domestic cats and pumas; 3 puma-to-puma transmissions in Florida were inferred. An additional 14 infections in pumas likely represented spillover events following contact with reservoir host domestic cat populations. Our data provide evidence that FeLV transmission from domestic cats to pumas occurs widely across the United States, and puma-to-puma transmission may occur in genetically and geographically constrained populations. IMPORTANCE Feline leukemia virus (FeLV) is a retrovirus that primarily affects domestic cats. Close interactions with domestic cats, including predation, can lead to the interspecific transmission of the virus to pumas, bobcats, or other feline species. Some infected individuals develop progressive infections, which are associated with clinical signs of disease and can result in mortality. Therefore, outbreaks of FeLV in wildlife, including the North American puma and the endangered Florida panther, are of high conservation concern. This work provides a greater understanding of the dynamics of the transmission of FeLV between domestic cats and wild felids and presents evidence of multiple spillover events and infections in all sampled populations. These findings highlight the concern for pathogen spillover from domestic animals to wildlife but also identify an opportunity to understand viral evolution following cross-species transmissions more broadly.

Apathogenic proxies for transmission dynamics of a fatal virus.

Identifying drivers of transmission-especially of emerging pathogens-is a formidable challenge for proactive disease management efforts. While close social interactions can be associated with microbial sharing between individuals, and thereby imply dynamics important for transmission, such associations can be obscured by the influences of factors such as shared diets or environments. Directly-transmitted viral agents, specifically those that are rapidly evolving such as many RNA viruses, can allow for high-resolution inference of transmission, and therefore hold promise for elucidating not only which individuals transmit to each other, but also drivers of those transmission events. Here, we tested a novel approach in the Florida panther, which is affected by several directly-transmitted feline retroviruses. We first inferred the transmission network for an apathogenic, directly-transmitted retrovirus, feline immunodeficiency virus (FIV), and then used exponential random graph models to determine drivers structuring this network. We then evaluated the utility of these drivers in predicting transmission of the analogously transmitted, pathogenic agent, feline leukemia virus (FeLV), and compared FIV-based predictions of outbreak dynamics against empirical FeLV outbreak data. FIV transmission was primarily driven by panther age class and distances between panther home range centroids. FIV-based modeling predicted FeLV dynamics similarly to common modeling approaches, but with evidence that FIV-based predictions captured the spatial structuring of the observed FeLV outbreak. While FIV-based predictions of FeLV transmission performed only marginally better than standard approaches, our results highlight the value of proactively identifying drivers of transmission-even based on analogously-transmitted, apathogenic agents-in order to predict transmission of emerging infectious agents. The identification of underlying drivers of transmission, such as through our workflow here, therefore holds promise for improving predictions of pathogen transmission in novel host populations, and could provide new strategies for proactive pathogen management in human and animal systems.

Protein expression profiling, in silico classification and pathway analysis of cariogenic bacteria Streptococcus mutans under bacitracin stress conditions.

Introduction. Streptococcus mutans is a cariogenic bacterium that causes dental caries as well as being implicated in other dental pathologies and infective endocarditis. Bacitracin is a bactericidal antibiotic that induces cell wall stress in Gram-positive bacteria.Gap Statement. S. mutans is among the most characterized Gram-positive bacteria. However, the transcriptome and proteome of S. mutans have received less attention, and they are actually key in understanding the pathogenesis of any bacteria. In this study, we extracted the whole proteome of S. mutans grown under bacitracin stress. Such a proteome is anticipated to offer deep insights related to physiological dynamic fluctuations and, consequently, it may provide 'proteomic signatures' to be identified as potential targets.Aim. The aim of the study is to explore the general stress response that S. mutans exhibits at the proteome level when cell wall stress is imposed on it.Methodology. A sub-MIC concentration of bacitracin was added to the growth media of S. mutans followed by whole-cell protein extraction. The proteome was then subjected to high-throughput proteomics analysis, i.e. liquid chromatography tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins obtained through LC-MS/MS underwent analyses such as gene ontology, KEGG (Kyoto Encyclopaedia of Genes and Genomes) and DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis, and STRING for functional annotation, pathway enrichment and protein-protein interaction (PPI) networks, respectively. These proteins were also categorized into functional classes using the PANTHER (Protein Annotation Through Evolutionary Relationship) classification system.Result. LC-MS/MS produced data from 321 identified proteins. From these, 41 and 30 were found to be significantly over- (≥2 fold change) and underexpressed (≤0.4 fold change), respectively. In the upregulated proteins we mostly observed sortases and proteins involved in the EPS biosynthesis pathway, whereas among the downregulated proteins the majority related to glycolysis.Conclusion. The sortase family of proteins appear to be potential targets because they regulate various virulence factors and therefore can be targeted to inhibit multiple virulence pathways simultaneously. This study offers an understanding of proteomic fluctuations in response to cell wall stress and can thus help in identifying key players mediating virulence.

Influence of Padina gymnospora on Apoptotic Proteins of Oral Cancer Cells-a Proteome-Wide Analysis.

Oral carcinoma is one of the most vicious forms of cancer with a very low survival rate, as its patients often respond poorly to conventional chemotherapy. Presently several researchers are attempting to pursue an alternative to this therapy using natural products. Considering the promising strategy and induction of apoptosis to target the cancer cells, we evaluated the influence of a seaweed Padina gymnospora (15 µg/ml and 20 µg/ml) in enhancing apoptosis of oral cancer cells (KB-CHR-8-5) after 24-h incubation. The morphological changes indicating apoptosis were primarily assessed using a light microscope after which the apoptosis was confirmed by performing AO/EB staining method. Subsequently, MMP and ROS levels in the cells were assessed using Rh 123 and DCFH-DA staining procedures, respectively. All the above tests confirmed the ability of P. gymnospora to accelerate apoptosis in the oral cancer cells. As a next step, wide proteome analysis was performed where the proteins from P. gymnospora-treated cells were separated using the 2D electrophoresis technique and compared with that of control cells to isolate the differentially expressed proteins. This procedure resulted in the isolation of 10 proteins which were identified using MALDI-TOF/TOF MS, which established that most of the isolated proteins were part of the apoptotic process of the cell. The proteins identified are part of huge and complex pathways where it gets linked with many more genes which are also associated with apoptosis. Bioinformatics of these identified proteins was analyzed using STRING and PANTHER databases. These proteins contribute to cell apoptosis by affecting various functions, biological processes, and the synthesis of cellular components. PANTHER also demonstrated that these proteins belong to the classes of proteins that take part in several vital pathways of the cell among which the apoptotic pathway is the predominant one.

Abbott® ID NOW™ COVID-19 rapid molecular assay versus Hologic® Panther Aptima™ SARS-CoV-2 assay in nasopharyngeal specimens: results from 1-year retrospective study in an emergency department.

We compared ID Now™ and Hologic® Panther Aptima™ for the detection of SARS-COV-2. ID Now™ showed a positive and negative percent agreement of 86.9% and 99.7% respectively. This facilitates faster clinical decision-making, along with the rapid implementation of infection control measures, and improvement of patient flow in the emergency department toward inpatient wards.