Heavy metals toxicity on epigenetic modifications in the pathogenesis of Alzheimer's disease (AD).

Alzheimer's disease (AD) is a progressive decline in cognitive ability and behavior which eventually disrupts daily activities. AD has no cure and the progression rate varies unlikely. Among various causative factors, heavy metals are reported to be a significant hazard in AD pathogenesis. Metal-induced neurodegeneration has been focused globally with thorough research to unravel the mechanistic insights in AD. Recently, heavy metals suggested to play an important role in epigenetic alterations which might provide evidential results on AD pathology. Epigenetic modifications are known to play towards novel therapeutic approaches in treating AD. Though many studies focus on epigenetics and heavy metal implications in AD, there is a lack of research on heavy metal influence on epigenetic toxicity in neurological disorders. The current review aims to elucidate the plausible role of cadmium (Cd), iron (Fe), arsenic (As), copper (Cu), and lithium (Li) metals on epigenetic factors and the increase in amyloid beta and tau phosphorylation in AD. Also, the review discusses the common methods of heavy metal detection to implicate in AD pathogenesis. Hence, from this review, we can extend the need for future research on identifying the mechanistic behavior of heavy metals on epigenetic toxicity and to develop diagnostic and therapeutic markers in AD.

Inorganic clay nanocomposite system for improved cholinesterase inhibition and brain pharmacokinetics of donepezil.

Objective: Brain drug delivery for effective treatment of neurodegenerative disorders is limited due to the selective permeability of blood brain barrier (BBB). During the past few years, development of novel delivery system has attracted considerable attention of formulation scientists to overcome the permeability limitation caused by BBB.Significance: Based on the outcomes of this study and taking into consideration of the unique characteristics of laponite, it can be further explored to deliver many other central nervous system acting drugs.Methods: In the present study, laponite (LAP) nanocomposites were exploited for the improved brain delivery of donepezil (DZ) following encapsulation approach due to their nano-size and positive charge at pH <9.Result: The size of prepared nanocomposites was 53.7 ± 4.0 to 137.7 ± 11.0 nm. The drug was released in a sustained manner till 120 h in phosphate buffer saline (pH 7.4) and acid phthalate buffer (pH 4.0). LAPDZ formulations inhibited acetylcholinesterase approximately by 82%, significantly higher (p < 0.05) than plain DZ (30%). Swiss albino mice exhibited enhanced brain uptake of LAPDZ administered via intravenous route. Promising pharmacokinetic parameters were observed in animals treated with LAPDZ. LAPDZ formulation showed half-life (t1/2), volume of distribution (Vd) and clearance (Cl) as 5.53 ± 0.40 h-1, 0.129 ± 0.02 L, 0.015 ± 0.002 L/h, respectively. While DZ solution showed the same parameters as 1.06 ± 0.12 h-1, 0.168 ± 0.01 L, 0.106 ± 0.013 L/h, respectively. The brain uptake of LAPDZ formulation was improved with quintuplet t1/2.Conclusion: Based on the results of present study, it is proposed that the formulated nanocomposite would result in improved patient compliance with therapeutic effect at lower doses.

CST, an Herbal Formula, Exerts Anti-Obesity Effects through Brain-Gut-Adipose Tissue Axis Modulation in High-Fat Diet Fed Mice.

The brain, gut, and adipose tissue interact to control metabolic pathways, and impairment in the brain-gut-adipose axis can lead to metabolic disorders, including obesity. Chowiseungcheng-tang (CST), a herbal formulation, is frequently used to treat metabolic disorders. Here, we investigated the anti-obesity effect of CST and its link with brain-gut-adipose axis using C57BL/6J mice as a model. The animals were provided with a normal research diet (NRD) or high-fat diet (HFD) in absence or presence of CST or orlistat (ORL) for 12 weeks. CST had a significant anti-obesity effect on a number of vital metabolic and obesity-related parameters in HFD-fed mice. CST significantly decreased the expression levels of genes encoding obesity-promoting neuropeptides (agouti-related peptide, neuropeptide Y), and increased the mRNA levels of obesity-suppressing neuropeptides (proopiomelanocortin, cocaine-and amphetamine-regulated transcript) in the hypothalamus. CST also effectively decreased the expression level of gene encoding obesity-promoting adipokine (retinol-binding protein-4) and increased the mRNA level of obesity-suppressing adipokine (adiponectin) in visceral adipose tissue (VAT). Additionally, CST altered the gut microbial composition in HFD groups, a phenomenon strongly associated with key metabolic parameters, neuropeptides, and adipokines. Our findings reveal that the anti-obesity impact of CST is mediated through modulation of metabolism-related neuropeptides, adipokines, and gut microbial composition.

Distribution and antimicrobial potential of endophytic fungi associated with ethnomedicinal plant Melastoma malabathricum L.

Distributions of endophytic fungi associated with ethnomedicinal plant Melastoma malabathricum L. was studied and 91 isolates belonging to 18 genera were recovered. The isolates were distributed to sordariomycetes (62.63%), dothideomycetes (19.78%), eurotiomycetes (7.69%), zygomycetes (4.19%), agaricomycetes (1.09%), and mycelia sterilia (4.39%). Based on colony morphology and examination of spores, the isolates were classified into 18 taxa, of which Colletotrichum, Phomopsis and Phoma were dominant, their relative frequencies were 23.07%, 17.58% and 12.08% respectively. The colonization rate of endophytic fungi was determined and found to be significantly higher in leaf segments (50.76%), followed by root (41.53%) and stem tissues (27.69%). All the isolates were screened for antimicrobial activity and revealed that 26.37% endophytic fungi were active against one or more pathogens. Twenty four isolates showing significant antimicrobial activity were identified by sequencing the ITS1-5.8S-ITS2 region of rRNA gene. Results indicated that endophytic fungi associated with leaf were functionally versatile as they showed antimicrobial activity against most of the tested pathogens. The endophytic fungi Diaporthe phaseolorum var. meridionalis (KF193982) inhibited all the tested bacterial pathogens, whereas, Penicillium chermesinum (KM405640) displayed most significant antifungal activity. This seems to be the first hand report to understand the distribution and antimicrobial ability of endophytic fungi from ethno-medicinal plant M. malabathricum.

Molecular insights and promise of oncolytic virus based immunotherapy.

Discovering a therapeutic that can counteract the aggressiveness of this disease's mechanism is crucial for improving survival rates for cancer patients and for better understanding the most different types of cancer. In recent years, using these viruses as an anticancer therapy has been thought to be successful. They mostly work by directly destroying cancer cells, activating the immune system to fight cancer, and expressing exogenous effector genes. For the treatment of tumors, oncolytic viruses (OVs), which can be modified to reproduce only in tumor tissues and lyse them while preserving the healthy non-neoplastic host cells and reinstating antitumor immunity which present a novel immunotherapeutic strategy. OVs can exist naturally or be created in a lab by altering existing viruses. These changes heralded the beginning of a new era of less harmful virus-based cancer therapy. We discuss three different types of oncolytic viruses that have already received regulatory approval to treat cancer as well as clinical research using oncolytic adenoviruses. The primary therapeutic applications, mechanism of action of oncolytic virus updates, future views of this therapy will be covered in this chapter.

Artificial intelligence and machine learning algorithms in the detection of heavy metals in water and wastewater: Methodological and ethical challenges.

Heavy metals (HMs) enter waterbodies through various means, which, when exceeding a threshold limit, cause toxic effects both on the environment and in humans upon entering their systems. Recent times have seen an increase in such HM influx incident rates. This requires an instant response in this regard to review the challenges in the available classical methods for HM detection and removal. As well as provide an opportunity to explore the applications of artificial intelligence (AI) and machine learning (ML) for the identification and further redemption of water and wastewater from the HMs. This review of research focuses on such applications in conjunction with the available in-silico models producing worldwide data for HM levels. Furthermore, the effect of HMs on various disease progressions has been provided, along with a brief account of prediction models analysing the health impact of HM intoxication. Also discussing the ethical and other challenges associated with the use of AI and ML in this field is the futuristic approach intended to follow, opening a wide scope of possibilities for improvement in wastewater treatment methodologies.

Microbiota-brain axis: Exploring the role of gut microbiota in psychiatric disorders - A comprehensive review.

Mental illness is a hidden epidemic in modern science that has gradually spread worldwide. According to estimates from the World Health Organization (WHO), approximately 10% of the world's population suffers from various mental diseases each year. Worldwide, financial and health burdens on society are increasing annually. Therefore, understanding the different factors that can influence mental illness is required to formulate novel and effective treatments and interventions to combat mental illness. Gut microbiota, consisting of diverse microbial communities residing in the gastrointestinal tract, exert profound effects on the central nervous system through the gut-brain axis. The gut-brain axis serves as a conduit for bidirectional communication between the two systems, enabling the gut microbiota to affect emotional and cognitive functions. Dysbiosis, or an imbalance in the gut microbiota, is associated with an increased susceptibility to mental health disorders and psychiatric illnesses. Gut microbiota is one of the most diverse and abundant groups of microbes that have been found to interact with the central nervous system and play important physiological functions in the human gut, thus greatly affecting the development of mental illnesses. The interaction between gut microbiota and mental health-related illnesses is a multifaceted and promising field of study. This review explores the mechanisms by which gut microbiota influences mental health, encompassing the modulation of neurotransmitter production, neuroinflammation, and integrity of the gut barrier. In addition, it emphasizes a thorough understanding of how the gut microbiome affects various psychiatric conditions.

Does gut brain axis has an impact on Parkinson's disease (PD)?

Parkinson's Disease (PD) is becoming a growing global concern by being the second most prevalent disease next to Alzheimer's Disease (AD). Henceforth new exploration is needed in search of new aspects towards the disease mechanism and origin. Evidence from recent studies has clearly stated the role of Gut Microbiota (GM) in the maintenance of the brain and as a root cause of various diseases and disorders including other neurological conditions. In the case of PD, with an unknown etiology, the GM is said to have a larger impact on the disease pathophysiology. Although GM and its metabolites are crucial for maintaining the normal physiology of the host, it is an undeniable fact that there is an influence of GM in the pathophysiology of PD. As such the Enteroendocrine Cells (EECs) in the epithelium of the intestine are one of the significant regulators of the gut-brain axis and act as a communication mediator between the gut and the brain. The communication is established via the molecules of neuroendocrine which are said to have a crucial part in neurological diseases such as AD, PD, and other psychiatry-related disorders. This review is focused on understanding the proper role of GM and EECs in PD. Here, we also focus on some of the metabolites and compounds that can interact with the PD genes causing various dysfunctions in the cell and facilitating the disease conditions using bioinformatical tools. Various mechanisms concerning EECs and PD, their identification, the latest studies, and available current therapies have also been discussed.

A comparative evaluation of prasugrel and clopidogrel in patients with acute coronary syndrome undergoing percutaneous coronary intervention.

OBJECTIVE: Primary objective of this study was to compare the efficacy of Prasugrel vs. Clopidogrel in the patients with acute coronary syndromes (ACS) undergoing percutaneous coronary intervention (PCI) by measuring inhibition of platelet aggregation after loading and maintenance dose of both the drugs. The patients were also assessed for safety of the drugs. METHODS: This was a randomised, double-blind, double-dummy, comparative, multicentric clinical trial in patients with acute coronary syndrome (unstable angina, non-ST elevation MI and ST elevation MI) undergoing PCI. The patients were randomly assigned to receive prasugrel (loading dose of 60 mg followed by maintenance dose of 10-mg once daily) or clopidogrel (loading dose of 300 mg followed by maintenance dose of 75 mg once daily) for a period of 12 weeks. All the patients were co-prescribed aspirin 325 mg with both the drugs. The primary efficacy end point in this study was percentage inhibition of ADP induced platelet aggregation (IPA) at 4 +/- 1 hours after the loading dose and at 30 +/- 3 days during maintenance treatment. The platelet aggregation of both the drugs was measured by whole blood aggregometer using 10 mmol of ADP as an aggregant. Though this study was not powered to see the difference in clinical efficacy parameters, the patients were observed for the incidence of nonfatal MI, nonfatal stroke, re-hospitalization, death, or need for urgent revascularization due to a cardiac ischemic event at days 30 and 90 during the study. The safety of study drugs were evaluated by incidence of major bleeding, reported adverse drug reaction and alterations of any laboratory parameters. RESULT: A total of 220 patients were enrolled at 11 centres across India. Ten patients were given the loading dose of prasugrel or clopidogrel but did not underwent PCI due to change in investigator's decision to go for PCI. Out of 210 eligible patients, 21 patients were discontinued during the study. 157 patients were evaluated for platelet inhibition after loading dose at 4 hours and 150 patients at day 30 during maintenance phase of antiplalelet therapy. The investigators could not perform this test in remaining patients due to urgency and criticality of the patients. 189 patients were observed for the incidence of nonfatal MI, nonfatal stroke, rehospitalisation, urgent revascularisation or death due to a cardiac ischemic event. All eligible patients who received at least a loading dose were evalauted for safety. In prasugrel group, 85 and 77 patients were evaluated for IPA at 4 hours and day 30 respectively whereas in clopdogrel group 72 and 73 patients were tested for IPA at 4 hours and at 30 days. Patients in prasugrel group have demonstrated significantly higher inhibition of platelets as compared to clopidogrel group (82.5% vs 71.1%) at 4 hours and at 30 days (84.1% vs 67.4%). The difference in inhibition of platelets between prasugrel and clopidogrel after loading dose and maintenenace dose was statistically significant (p < or = 0.01). The patients were also evaluated for drug hyporesponsiveness to antiplatelet therapy if IPA was < 20% at day 30 from the baseline. More patients on prasugrel have shown response to antiplatlet therapy than on clopidogrel (97.4% vs 87.6%). The difference between the two groups was statistically significant (p < 0.05). There was no difference observed during the study in the incidence of nonfatal MI, nonfatal stroke, death, rehospitalisation or need for urgent revascularisation due to a cardiac event between prasugrel and clopidogrel. Both the drugs were found to be to be well tolerated and have comparable safety profile. CONCLUSION: This study suggests that prasugrel is more effective than clopidogrel as an anti platelet drug as evident by inhibition of platelet aggregation. More patients on clopidogrel are likely to have poor response to therapy as compared to prasugrel. Both the drugs were well tolerated and have comparable safety profile.

Outpatient Clinical Markers for Laryngopharyngeal Reflux Disease: A Correlational Study.

Laryngopharyngeal reflux disease (LPRD) is the result of retrograde flow of gastric contents to the laryngopharynx which comes in contact with tissues of the upper aerodigestive tract. Due to ill defined criteria for diagnosis & followup, LPRD patients are underdiagnosed & undertreated. Reflux Symptom Index (RSI) and the Reflux Finding Score (RFS) are two clinical methods which can be utilised especially in the outpatient setup. This study was done with the aim to assess various laryngoscopic findings in patients with LPRD diagnosed symptomatically and examine the correlation between the RSI & RFS by comparing these two indices. This prospective analytical study was conducted at a tertiary care centre in Bangalore in the Department of ENT for a period of 24 months between Dec 2020 to Dec 2022. The study included patients aged 18 to 60 years diagnosed with LPRD based on symptoms as per RSI score (> 13). RSI & RFS were assessed on diagnosis and patients were followed up for 1, 3 & 6 months for assessment. Total 96 patients were enrolled, with mean age of be 42.49 ± 11.33 years. Prevalence was found to be more in females (61.5%). The most common symptom according to RSI was frequent throat clearing & globus sensation (sensation of something sticking in throat) and most common finding according to RFS was erythema/hyperemia. The mean score of RSI and RFI was found to reduce with treatment at different intervals in follow-up visits. There was a significant strength of association between the RSI and RFS at baseline, 1st month, 3rd month and 6th month of follow-up (r = 0.568, r = 0.684, r = 0.774, r = 0.736 respectively) (p < 0.001).The RFS and RSI showed statistically significant strong relationships between total scores and sign and symptom characteristics. On follow-up, there was a significant reduction in the RSI which was also correlated with a reduction in RFS.

Association of Biochemical Parameters and Screening for Mutations in the MCU Gene in Alzheimer's Disease Patients.

The most prevalent form of dementia, Alzheimer's disease (AD) is a chronic illness that is on the rise among the geriatric population. Even though research into its biochemical, genetic, and cytogenetic pathways has advanced, its aetiology is still unclear and complex. In this study, we recruited sixty-eight participants diagnosed with AD where the cytogenetic, biochemical parameters and genetic mutations were analysed. Our results revealed chromosomal aberrations such as aneuploidies in the peripheral blood of Alzheimer's disease patients. Biochemical parameters revealed no statistical significance in the study though a pattern could be observed in the serum levels. Further few novel mutations at the c.21 C > T, c.56G > A were observed in the MCU gene of mitochondrial calcium uniporter. All these findings reveal the need for a larger cohort study to gain a better and more detailed understanding of the aetiology of Alzheimer's disease.

A cooperativity between virus and bacteria during respiratory infections.

Respiratory tract infections remain the leading cause of morbidity and mortality worldwide. The burden is further increased by polymicrobial infection or viral and bacterial co-infection, often exacerbating the existing condition. Way back in 1918, high morbidity due to secondary pneumonia caused by bacterial infection was known, and a similar phenomenon was observed during the recent COVID-19 pandemic in which secondary bacterial infection worsens the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) condition. It has been observed that viruses paved the way for subsequent bacterial infection; similarly, bacteria have also been found to aid in viral infection. Viruses elevate bacterial infection by impairing the host's immune response, disrupting epithelial barrier integrity, expression of surface receptors and adhesion proteins, direct binding of virus to bacteria, altering nutritional immunity, and effecting the bacterial biofilm. Similarly, the bacteria enhance viral infection by altering the host's immune response, up-regulation of adhesion proteins, and activation of viral proteins. During co-infection, respiratory bacterial and viral pathogens were found to adapt and co-exist in the airways of their survival and to benefit from each other, i.e., there is a cooperative existence between the two. This review comprehensively reviews the mechanisms involved in the synergistic/cooperativity relationship between viruses and bacteria and their interaction in clinically relevant respiratory infections.

Effect of 5-azacytidine on in vitro biofilm formation of Streptococcus pneumoniae.

Streptococcus pneumoniae is a gram-positive bacterium that causes otitis media, pneumonia, meningitis and sepsis in young children and the elderly. Previous studies reported that pneumococci in different diseases do not behave as planktonic cells, but predominantly show characteristics of a biofilm. In this study we examine the effect of 5-azacytidine on S. pneumoniae, particularly on biofilm formation and investigate the gene expression involved in synthesis of autoinducer-2, competence and DNA repair. The effect of 5-aza on in vitro biofilm formation was studied by the crystal violet microtiter plate method. The S. pneumoniae biofilms were grown with different concentration of 5-azacytidine (15-500 µm), at variable time intervals and the inhibition percentages were calculated. The effects of 5-aza on the morphology of biofilms were analyzed by scanning electron microscope. The relative quantification of 11 genes of biofilms grown with 5-aza involved in autoinducer-2 synthesis, competence and DNA repair was carried out by real-time RT-PCR with respect to biofilms grown without 5-aza. The crystal violet microtiter assay detected a significant inhibitory effect of 5-aza on in vitro biofilm formation, at concentration that did not inhibited planktonic cell growth. The SEM analysis demonstrated thin and disrupted biofilms, without micro-colonies in the samples treated with 5-aza, while these structures were present in the biofilms grown without 5-aza. The relative quantification of gene expression of 5-aza biofilms showed a significant down regulation of genes involved in the methionine and homocysteine recycling pathway which produces quorum sensing molecule autoinducer-2 as by-products. A significant decrease in the expressions of luxS, metK, pfs and cmK was detected. In conclusion, 5-aza inhibits in vitro biofilm formation and decreases the expression of luxS, pfs and metK, which are involved in the synthesis of autoinducer-2 as by-products of the methionine recycling pathway. The inhibitory effect of 5-aza may be either due to down regulation of pfs, luxS and metK or due to accumulation of the toxic substrate of pfs, luxS and metK genes.

Gene expression profile of early in vitro biofilms of Streptococcus pneumoniae.

In this study, the gene expression profile of early in vitro Streptococcus pneumoniae biofilm with respect to planktonic cells in cDNA microarray analysis is reported. Microarray analysis with respect to planktonic cells was performed on total RNA extracted from biofilms grown in 24-well microtiter plates. To validate the microarray results, real-time RT-PCR was performed on 13 differentially expressed genes and one constitutively expressed gene. The cDNA-microarray analyses identified 89 genes that were significantly differentially expressed in biofilm and planktonic cells. Genes involved in isoprenoid biosynthesis, cell wall biosynthesis, translation and purine and pyrimidine nucleotide metabolic pathways were exclusively expressed in the biofilms, whereas transcription regulator genes were exclusively expressed in planktonic cells. The real-time RT-PCR results of 13 differentially regulated genes were completely in agreement with the microarray data. The exclusive up regulation in biofilms of genes involved in the mevalonate pathway, cell wall biosynthesis, translation and purine and pyrimidine nucleotide metabolic pathways suggests that expression of these genes may be required for initial biofilm formation, and growth and survival of bacteria in biofilms. The up regulation of related genes suggests that cells in biofilms may be under stress conditions and possibly actively involved in the protein synthesis required to adapt to a new environment.

Revisiting the role of cyanobacteria-derived metabolites as antimicrobial agent: A 21st century perspective.

Cyanobacterial species are ancient photodiazotrophs prevalent in freshwater bodies and a natural reservoir of many metabolites (low to high molecular weight) such as non-ribosomal peptides, polyketides, ribosomal peptides, alkaloids, cyanotoxins, and isoprenoids with a well-established bioactivity potential. These metabolites enable cyanobacterial survival in extreme environments such as high salinity, heavy metals, cold, UV-B, etc. Recently, these metabolites are gaining the attention of researchers across the globe because of their tremendous applications as antimicrobial agents. Many reports claim the antimicrobial nature of these metabolites; unfortunately, the mode of action of such metabolites is not well understood and/or known limited. Henceforth, this review focuses on the properties and potential application, also critically highlighting the possible mechanism of action of these metabolites to offer further translational research. The review also aims to provide a comprehensive insight into current gaps in research on cyanobacterial biology as antimicrobials and hopes to shed light on the importance of continuing research on cyanobacteria metabolites in the search for novel antimicrobials.

Real-time PCR assay based on topoisomerase-II gene for detection of Fusarium udum.

Fusarium wilt is an important soilborne disease of pigeonpea, caused by Fusarium udum. In this study, we have designed a real-time PCR assay for the detection of Fusarium udum from infected pigeonpea plants. Based on Topoisomerase-II gene sequence data from Fusarium udum and other related Fusarium species, a pair of primer was designed. The species-specific primers were tested in real-time PCR SYBR green assay. No increasing fluorescence signals exceeding the baseline threshold was observed with tested microbes, except Fusarium udum DNA. A single dissociation peak of increased fluorescence was obtained for the specific primers at melting temperature of 81.25°C. The real-time PCR showed a lowest detection of 0.1 pg genomic DNA. The assay was more sensitive, accurate and less time consuming for detection of Fusarium udum in infected plants root.

Antimicrobial sensitivity profiling of bacterial communities recovered from effluents of municipal solid waste dumping site.

The diversity of antibiotic-resistance bacteria (ARB) from the effluents of Aizawl city municipal waste dumping site was studied using a culture-dependent method. The present study molecularly identified 73 isolates that were differentiated into three phyla (Proteobacteria, Actinobacteria, and Firmicutes) belonging to 17 family and 22 genera. Bacillus constitutes the most dominant genus comprising 16% of the total isolates. The topology of the phylogenetic tree differentiates them into five major clades. Corynebacterium and Rhodococcus which are morphologically alike were clustered together and the Gram-positive bacteria such as Staphylococcus, Bacillus, and Lysinibacillus formed a separate cluster. Antibiotic resistance of the identified bacterial isolates was performed using 19 different antibiotics. Among the isolates, 70 of them found resistant to polymixin B and nalidixic acid and 10 isolates exhibited resistance to 15 tested antibiotics. The present study revealed that bacteria with antibiotic resistance are extensively distributed in the effluents of the dumping site and may serve as a significant reservoir for the spreading of antibiotic resistance to opportunistic pathogens.

Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review.

Plant viral diseases are the foremost threat to sustainable agriculture, leading to several billion dollars in losses every year. Many viruses infecting several crops have been described in the literature; however, new infectious viruses are emerging frequently through outbreaks. For the effective treatment and prevention of viral diseases, there is great demand for new techniques that can provide accurate identification on the causative agents. With the advancements in biochemical and molecular biology techniques, several diagnostic methods with improved sensitivity and specificity for the detection of prevalent and/or unknown plant viruses are being continuously developed. Currently, serological and nucleic acid methods are the most widely used for plant viral diagnosis. Nucleic acid-based techniques that amplify target DNA/RNA have been evolved with many variants. However, there is growing interest in developing techniques that can be based in real-time and thus facilitate in-field diagnosis. Next-generation sequencing (NGS)-based innovative methods have shown great potential to detect multiple viruses simultaneously; however, such techniques are in the preliminary stages in plant viral disease diagnostics. This review discusses the recent progress in the use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases. New portable devices and technologies that could provide real-time analyses in a relatively short period of time are prime important for in-field diagnostics. Current development and application of such tools and techniques along with their potential limitations in plant virology are likewise discussed in detail.

Urban Particles Elevated Streptococcus pneumoniae Biofilms, Colonization of the Human Middle Ear Epithelial Cells, Mouse Nasopharynx and Transit to the Middle Ear and Lungs.

Air-pollutants containing toxic particulate matters (PM) deposit in the respiratory tract and increases microbial infections. However, the mechanism by which this occurs is not well understood. This study evaluated the effect of urban particles (UP) on Streptococcus pneumoniae (pneumococcus) in vitro biofilm formation, colonization of human middle ear epithelium cells (HMEECs) as well as mouse nasal cavity and its transition to the middle ear and lungs. The in vitro biofilms and planktonic growth of S. pneumoniae were evaluated in metal ion free medium in the presence of UP. Biofilms were quantified by crystal violet (CV) microplate assay, colony forming unit (cfu) counts and resazurin staining. Biofilm structures were analyzed using a scanning electron microscope (SEM) and confocal microscopy (CM). Gene expressions of biofilms were evaluated using real time RT-PCR. Effects of UP exposure on S. pneumoniae colonization to HMEECs were evaluated using fluorescent in-situ hybridization (FISH), cell viability was detected using the Ezcyto kit, apoptosis in HMEECs were evaluated using Annexin-V/PI based cytometry analysis and reactive oxygen species (ROS) production were evaluated using the Oxiselect kit. Alteration of HMEECs gene expressions on UP exposure or pneumococci colonization was evaluated using microarray. In vivo colonization of pneumococci in the presence of UP and transition to middle ear and lungs were evaluated using an intranasal mice colonization model. The UP exposure significantly increased (*p < 0.05) pneumococcal in vitro biofilms and planktonic growth. In the presence of UP, pneumococci formed organized biofilms with a matrix, while in absence of UP bacteria were unable to form biofilms. The luxS, ply, lytA, comA, comB and ciaR genes involved in bacterial pathogenesis, biofilm formation and quorum sensing were up-regulated in pneumococci biofilms grown in the presence of UP. The HMEECs viability was significantly decreased (p < 0.05) and bacteria colonization was significantly elevated (p < 0.05) in co-treatment (UP + S. pneumoniae) when compared to single treatment. Similarly, increased apoptosis and ROS production were detected in HMEECs treated with UP + pneumococci. The microarray analysis of HMEECs revealed that the genes involve in apoptosis and cell death, inflammation, and immune response, were up-regulated in co-treatment and were unchanged or expressed in less fold in single treatments of UP or S. pneumoniae. The in vivo study showed an increased pneumococcal colonization of the nasopharynx in the presence of UP and a higher transition of bacteria to the middle ear and lungs in the presence of UP. The UP exposure elevated S. pneumoniae in vitro biofilm and colonization of HMEECs, and in vivo mouse nasopharyngeal colonization, and increased dissemination to mouse middle ear and lungs.