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18 pages, 4559 KiB

Open AccessArticle

MicroRNA Targets PAP1 to Mediate Melanization in Plutella xylostella (Linnaeus) Infected by Metarhizium anisopliae

by Zhantao Zhang, Fengliang Jin, Junlin Huang, Surajit De Mandal, Lu Zeng, Junaid Zafar and Xiaoxia Xu

Int. J. Mol. Sci. 2024, 25(2), 1140; https://doi.org/10.3390/ijms25021140 - 17 Jan 2024

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Abstract

MicroRNAs (miRNAs) play a pivotal role in important biological processes by regulating post-transcriptional gene expression and exhibit differential expression patterns during development, immune responses, and stress challenges. The diamondback moth causes significant economic damage to crops worldwide. Despite substantial advancements in understanding the [...] Read more.

MicroRNAs (miRNAs) play a pivotal role in important biological processes by regulating post-transcriptional gene expression and exhibit differential expression patterns during development, immune responses, and stress challenges. The diamondback moth causes significant economic damage to crops worldwide. Despite substantial advancements in understanding the molecular biology of this pest, our knowledge regarding the role of miRNAs in regulating key immunity-related genes remains limited. In this study, we leveraged whole transcriptome resequencing data from Plutella xylostella infected with Metarhizium anisopliae to identify specific miRNAs targeting the prophenoloxidase-activating protease1 (PAP1) gene and regulate phenoloxidase (PO) cascade during melanization. Seven miRNAs (pxy-miR-375-5p, pxy-miR-4448-3p, pxy-miR-279a-3p, pxy-miR-3286-3p, pxy-miR-965-5p, pxy-miR-8799-3p, and pxy-miR-14b-5p) were screened. Luciferase reporter assays confirmed that pxy-miR-279a-3p binds to the open reading frame (ORF) and pxy-miR-965-5p to the 3′ untranslated region (3′ UTR) of PAP1. Our experiments demonstrated that a pxy-miR-965-5p mimic significantly reduced PAP1 expression in P. xylostella larvae, suppressed PO activity, and increased larval mortality rate. Conversely, the injection of pxy-miR-965-5p inhibitor could increase PAP1 expression and PO activity while decreasing larval mortality rate. Furthermore, we identified four LncRNAs (MSTRG.32910.1, MSTRG.7100.1, MSTRG.6802.1, and MSTRG.22113.1) that potentially interact with pxy-miR-965-5p. Interference assays using antisense oligonucleotides (ASOs) revealed that silencing MSTRG.7100.1 and MSTRG.22113.1 increased the expression of pxy-miR-965-5p. These findings shed light on the potential role of pxy-miR-965-5p in the immune response of P. xylostella to M. anisopliae infection and provide a theoretical basis for biological control strategies targeting the immune system of this pest. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections 2.0)

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11 pages, 1383 KiB

Open AccessArticle

In Vitro and In Vivo Antimicrobial Activities of Vancomycin and Rifampin against Elizabethkingia anophelis

by I-Fan Lin, Chung-Hsu Lai, Shang-Yi Lin, Ching-Chi Lee, Nan-Yao Lee, Po-Yu Liu, Chih-Hui Yang, Yi-Han Huang and Jiun-Nong Lin

Int. J. Mol. Sci. 2023, 24(23), 17012; https://doi.org/10.3390/ijms242317012 - 30 Nov 2023

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Abstract

Elizabethkingia anophelis has emerged as a critical human pathogen, and a number of isolated reports have described the successful treatment of Elizabethkingia infections with vancomycin, a drug that is typically used to target Gram-positive bacteria. This study employed in vitro broth microdilution checkerboard and [...] Read more.

Elizabethkingia anophelis has emerged as a critical human pathogen, and a number of isolated reports have described the successful treatment of Elizabethkingia infections with vancomycin, a drug that is typically used to target Gram-positive bacteria. This study employed in vitro broth microdilution checkerboard and time-kill assays, as well as in vivo zebrafish animal models to evaluate the individual and combination antimicrobial effects of vancomycin and rifampin against E. anophelis. The minimum inhibitory concentration ranges of vancomycin and rifampin against 167 isolates of E. anophelis were 16–256 mg/L and 0.06–128 mg/L, respectively. The checkerboard assay results revealed a synergistic effect between vancomycin and rifampin in 16.8% (28/167) of the isolates. Time-kill assays were implemented for 66 isolates, and the two-drug combination had a synergistic interaction in 57 (86.4%) isolates. In vivo zebrafish studies revealed that treatment with vancomycin monotherapy, rifampin monotherapy, or vancomycin–rifampin combination therapy yielded a higher survival rate than the control group treatment with 0.9% saline. The results of this study support the use of vancomycin to treat E. anophelis infections. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections 2.0)

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18 pages, 3502 KiB

Open AccessArticle

Metabolome Profiling in the Plasma of Dogs with Idiopathic Dilated Cardiomyopathy: A Multiplatform Mass-Spectrometry-Based Approach

by Ivana Rubić, Stefan Weidt, Richard Burchmore, Alan Kovačević, Josipa Kuleš, Peter David Eckersall, Marin Torti, Ines Jović, Mislav Kovačić, Jelena Gotić, Renata Barić Rafaj, Predrag Novak, Marko Samardžija and Vladimir Mrljak

Int. J. Mol. Sci. 2023, 24(20), 15182; https://doi.org/10.3390/ijms242015182 - 14 Oct 2023

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Abstract

Dilated cardiomyopathy is one of the important diseases in dogs and humans. The second most common cause of heart failure in dogs is idiopathic dilated cardiomyopathy (iDCM), which results in heart failure or sudden cardiac death due to arrhythmia. This study aimed to [...] Read more.

Dilated cardiomyopathy is one of the important diseases in dogs and humans. The second most common cause of heart failure in dogs is idiopathic dilated cardiomyopathy (iDCM), which results in heart failure or sudden cardiac death due to arrhythmia. This study aimed to determine changes in the plasma metabolome of dogs with iDCM compared to healthy dogs. For that purpose, a multiplatform mass-spectrometry-based approach was used. In this study, we included two groups of dogs: 12 dogs with iDCM and 8 healthy dogs. A total of 272 metabolites were detected in the plasma samples of dogs by combining three approaches but four MS-based platforms (GC-MS, LC-MS (untargeted), LC-MS (targeted), and FIA-MS (targeted) methods). Our findings demonstrated changes in the canine plasma metabolome involved in the development of iDCM, including the different concentrations of amino acids, biogenic amines, acylcarnitines, triglycerides and diglycerides, sphingomyelins, and organic acids. The results of this study will enable the detection and monitoring of pathophysiological mechanisms involved in the development of iDCM in the future. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections 2.0)

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15 pages, 292 KiB

Open AccessArticle

The Essential Role of PCR and PCR Panel Size in Comparison with Urine Culture in Identification of Polymicrobial and Fastidious Organisms in Patients with Complicated Urinary Tract Infections

by Xingpei Hao, Marcus Cognetti, Chiraag Patel, Nathalie Jean-Charles, Arun Tumati, Rhonda Burch-Smith, Mara Holton and Deepak A. Kapoor

Int. J. Mol. Sci. 2023, 24(18), 14269; https://doi.org/10.3390/ijms241814269 - 19 Sep 2023

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Abstract

Complicated urinary tract infections (cUTIs) are difficult to treat, consume substantial resources, and cause increased patient morbidity. Data suggest that cUTI may be caused by polymicrobial and fastidious organisms (PMOs and FOs, respectively); as such, urine culture (UC) may be an unreliable diagnostic [...] Read more.

Complicated urinary tract infections (cUTIs) are difficult to treat, consume substantial resources, and cause increased patient morbidity. Data suggest that cUTI may be caused by polymicrobial and fastidious organisms (PMOs and FOs, respectively); as such, urine culture (UC) may be an unreliable diagnostic tool for detecting cUTIs. We sought to determine the utility of PCR testing for patients presumed to have a cUTI and determine the impact of PCR panel size on organism detection. We reviewed 36,586 specimens from patients with presumptive cUTIs who received both UC and PCR testing. Overall positivity rate for PCR and UC was 52.3% and 33.9%, respectively (p < 0.01). PCR detected more PMO and FO than UC (PMO: 46.2% vs. 3.6%; FO: 31.3% vs. 0.7%, respectively, both p < 0.01). Line-item concordance showed that PCR detected 90.2% of organisms identified by UC whereas UC discovered 31.9% of organisms detected by PCR (p < 0.01). Organism detection increased with expansion in PCR panel size from 5–25 organisms (p < 0.01). Our data show that overall positivity rate and the detection of individual organisms, PMO and FO are significantly with PCR testing and that these advantages are ideally realized with a PCR panel size of 25 or greater. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections 2.0)

20 pages, 4461 KiB

Open AccessArticle

The Molecular Mechanisms Employed by the Parasite Myxobolus bejeranoi (Cnidaria: Myxozoa) from Invasion through Sporulation for Successful Proliferation in Its Fish Host

by Keren Maor-Landaw, Itamar Avidor, Nadav Rostowsky, Barbara Salti, Margarita Smirnov, Maya Ofek-Lalzar, Liron Levin, Vera Brekhman and Tamar Lotan

Int. J. Mol. Sci. 2023, 24(16), 12824; https://doi.org/10.3390/ijms241612824 - 15 Aug 2023

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Abstract

Myxozoa is a unique group of obligate endoparasites in the phylum Cnidaria that can cause emerging diseases in wild and cultured fish populations. Recently, we identified a new myxozoan species, Myxobolus bejeranoi, which infects the gills of cultured tilapia while suppressing host [...] Read more.

Myxozoa is a unique group of obligate endoparasites in the phylum Cnidaria that can cause emerging diseases in wild and cultured fish populations. Recently, we identified a new myxozoan species, Myxobolus bejeranoi, which infects the gills of cultured tilapia while suppressing host immunity. To uncover the molecular mechanisms underlying this successful parasitic strategy, we conducted transcriptomics analysis of M. bejeranoi throughout the infection. Our results show that histones, which are essential for accelerated cell division, are highly expressed even one day after invasion. As the infection progressed, conserved parasitic genes that are known to modulate the host immune reaction in different parasitic taxa were upregulated. These genes included energy-related glycolytic enzymes, as well as calreticulin, proteases, and miRNA biogenesis proteins. Interestingly, myxozoan calreticulin formed a distinct phylogenetic clade apart from other cnidarians, suggesting a possible function in parasite pathogenesis. Sporogenesis was in its final stages 20 days post-exposure, as spore-specific markers were highly expressed. Lastly, we provide the first catalog of transcription factors in a Myxozoa species, which is minimized compared to free-living cnidarians and is dominated by homeodomain types. Overall, these molecular insights into myxozoan infection support the concept that parasitic strategies are a result of convergent evolution. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections 2.0)

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10 pages, 767 KiB

Open AccessArticle

Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples

by Claudio Neidhöfer, Maria Bagniceva, Nina Wetzig, Martin A. Sieber, Ralf Thiele and Marijo Parčina

Int. J. Mol. Sci. 2023, 24(14), 11262; https://doi.org/10.3390/ijms241411262 - 09 Jul 2023

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Abstract

Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking [...] Read more.

Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections 2.0)

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Review

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12 pages, 266 KiB

Open AccessReview

Application of Advanced Molecular Methods to Study Early-Onset Neonatal Sepsis

by Chrysoula Kosmeri, Vasileios Giapros, Anastasios Serbis and Maria Baltogianni

Int. J. Mol. Sci. 2024, 25(4), 2258; https://doi.org/10.3390/ijms25042258 - 13 Feb 2024

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Abstract

Early-onset sepsis (EOS) is a global health issue, considered one of the primary causes of neonatal mortality. Diagnosis of EOS is challenging because its clinical signs are nonspecific, and blood culture, which is the current gold-standard diagnostic tool, has low sensitivity. Commonly used [...] Read more.

Early-onset sepsis (EOS) is a global health issue, considered one of the primary causes of neonatal mortality. Diagnosis of EOS is challenging because its clinical signs are nonspecific, and blood culture, which is the current gold-standard diagnostic tool, has low sensitivity. Commonly used biomarkers for sepsis diagnosis, including C-reactive protein, procalcitonin, and interleukin-6, lack specificity for infection. Due to the disadvantages of blood culture and other common biomarkers, ongoing efforts are directed towards identifying innovative molecular approaches to diagnose neonates at risk of sepsis. This review aims to gather knowledge and recent research on these emerging molecular methods. PCR-based techniques and unrestricted techniques based on 16S rRNA sequencing and 16S–23S rRNA gene interspace region sequencing offer several advantages. Despite their potential, these approaches are not able to replace blood cultures due to several limitations; however, they may prove valuable as complementary tests in neonatal sepsis diagnosis. Several microRNAs have been evaluated and have been proposed as diagnostic biomarkers in EOS. T2 magnetic resonance and bioinformatic analysis have proposed potential biomarkers of neonatal sepsis, though further studies are essential to validate these findings. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections 2.0)

22 pages, 5276 KiB

Open AccessReview

The Immune Response to the Myxozoan Parasite Myxobolus cerebralis in Salmonids: A Review on Whirling Disease

by Naveed Akram, Mansour El-Matbouli and Mona Saleh

Int. J. Mol. Sci. 2023, 24(24), 17392; https://doi.org/10.3390/ijms242417392 - 12 Dec 2023

Viewed by 1027

Abstract

Salmonids are affected by the economically significant whirling disease (WD) caused by the myxozoan parasite Myxobolus cerebralis. In the past, it was endemic to Eurasia, but it has now spread to different regions of North America, Europe, New Zealand, and South Africa. [...] Read more.

Salmonids are affected by the economically significant whirling disease (WD) caused by the myxozoan parasite Myxobolus cerebralis. In the past, it was endemic to Eurasia, but it has now spread to different regions of North America, Europe, New Zealand, and South Africa. Among salmonids, rainbow trout is considered the most highly susceptible host. Upon entering to the host’s body, the parasite invades the spine and cranium, resulting in whirling behaviour, a blackened tail, and destruction of cartilage. The disease is characterized by the infiltration of numerous inflammatory cells, primarily lymphocytes and macrophages, with the onset of fibrous tissue infiltration. Several efforts have been undertaken to investigate the role of various immune modulatory molecules and immune regulatory genes using advanced molecular methods including flow cytometry and transcriptional techniques. Investigation of the molecular and cellular responses, the role of STAT3 in Th17 cell differentiation, and the inhibitory actions of suppressors of cytokine signaling (SOCS) on interferons and interleukins, as well as the role of natural resistance-associated macrophage proteins (Nramp) in WD have significantly contributed to our understanding of the immune regulation mechanism in salmonids against M. cerebralis. This review thoroughly highlights previous research and discusses potential future directions for understanding the molecular immune response of salmonids and the possible development of prophylactic approaches against WD. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections 2.0)

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