Polymerase Chain Reaction Sequencing Research Articles

Outcomes in solid organ transplant recipients receiving organs from a donor with Fusarium solani species complex meningitis.

Five organs (heart, right lung, liver, right, and left kidneys) from a deceased patient were transplanted into five recipients in four US states; the deceased patient was identified as part of a healthcare-associated fungal meningitis outbreak among patients who underwent epidural anesthesia in Matamoros, Mexico. After transplant surgeries occurred, Fusarium solani species complex, a fungal pathogen with a high case-mortality rate, was identified in cerebrospinal fluid from the organ donor by metagenomic next-generation sequencing (mNGS) and fungal-specific polymerase chain reaction and in plasma by mNGS. Four of five transplant recipients received recommended voriconazole prophylaxis; four were monitored weekly by serum (1-3)-β-d-glucan testing. All five were monitored for signs of infection for at least 3 months following transplantation. The liver recipient had graft failure, which was attributed to an etiology unrelated to fungal infection. No fungal DNA was identified in sections of the explanted liver, suggesting that F. solani species complex did not contribute to graft failure. The remaining recipients experienced no signs or symptoms suggestive of fusariosis. Antifungal prophylaxis may be useful in preventing donor-derived infections in recipients of organs from donors that are found to have Fusarium meningitis.

Status of Cassava Witches’ Broom Disease in the Philippines and Identification of Potential Pathogens by Metagenomic Analysis

Cassava witches’ broom disease (CWBD) is one of the most devastating diseases of cassava (Manihot esculenta Crantz), and it threatens global production of the crop. In 2017, a phytoplasma, Candidatus Phytoplasma luffae (Ca. P. luffae), was reported in the Philippines, and it has been considered as the causal agent, despite unknown etiology and transmission of CWBD. In this study, the nationwide occurrence of CWBD was assessed, and detection of CWBD’s pathogen was attempted using polymerase chain reaction (PCR) and next-generation sequencing (NGS) techniques. The results showed that CWBD has spread and become severe, exhibiting symptoms such as small leaf proliferation, shortened internodes, and vascular necrosis. PCR analysis revealed a low phytoplasma detection rate, possibly due to low titer, uneven distribution, or absence in the CWBD-symptomatic cassava. In addition, NGS techniques confirm the PCR results, revealing the absence or extremely low phytoplasma read counts, but a surprisingly high abundance of fastidious and xylem-limited fungus, Ceratobasidium sp. in CWBD-symptomatic plants. These findings cast doubt over the involvement of phytoplasma in CWBD and instead highlight the potential association of Ceratobasidium sp., strongly supporting the recent findings in mainland Southeast Asia. Further investigations are needed to verify the etiology of CWBD and identify infection mechanisms of Ceratobasidium sp. to develop effective diagnostic and control methods for disease management.

Exosomes derived from human urine–derived stem cells ameliorate IL-1β-induced intervertebral disk degeneration

BackgroundHuman intervertebral disk degeneration (IVDD) is a sophisticated degenerative pathological process. A key cause of IVDD progression is nucleus pulposus cell (NPC) degeneration, which contributes to excessive endoplasmic reticulum stress in the intervertebral disk. However, the mechanisms underlying IVDD and NPC degeneration remain unclear.MethodsWe used interleukin (IL)-1β stimulation to establish an NPC-degenerated IVDD model and investigated whether human urine–derived stem cell (USC) exosomes could prevent IL-1β-induced NPC degeneration using western blotting, quantitative real-time polymerase chain reaction, flow cytometry, and transcriptome sequencing techniques.ResultsWe successfully extracted and identified USCs and exosomes from human urine. IL-1β substantially downregulated NPC viability and induced NPC degeneration while modulating the expression of SOX-9, collagen II, and aggrecan. Exosomes from USCs could rescue IL-1β-induced NPC degeneration and restore the expression levels of SOX-9, collagen II, and aggrecan.ConclusionsUSC-derived exosomes can prevent NPCs from degeneration following IL-1β stimulation. This finding can aid the development of a potential treatment strategy for IVDD.

Integrating the Idylla™ System Alongside a Real-Time Polymerase Chain Reaction and Next-Generation Sequencing for Investigating Gene Fusions in Pleural Effusions from Non-Small-Cell Lung Cancer Patients: A Pilot Study

Integrating the Idylla™ System Alongside a Real-Time Polymerase Chain Reaction and Next-Generation Sequencing for Investigating Gene Fusions in Pleural Effusions from Non-Small-Cell Lung Cancer Patients: A Pilot Study

Using rDNA ITS2 barcoding to identify kratom (Mitragyna speciosa) from the genus Mitragyna and Neolamarckia cadamba.

This study collected 80 samples of suspected kratom plant powder. A polymerase chain reaction sequence analysis was conducted using two sets of DNA barcode primers for plant ribosomal (r)DNA internal transcribed spacers (ITSs), namely, ITS3/ITS4 and ITS-p3/ITS-u4. Among the 80 samples, 40 were analyzed using the ITS3/ITS4 primer pair, and then DNA sequences were subjected to a National Center for Biotechnology Information-Basic Local Alignment Search Tool (NCBI-BLAST) comparison. Results showed that 29 samples had a 100% match (364/364) with Mitragyna speciosa (kratom), and 6 samples had a 99.73% match (363/364) with M. speciosa, whereas 5 samples had disordered and unreadable sequences. The 5 unreadable samples and an additional 40 suspected kratom samples were then analyzed using the ITS-p3/ITS-u4 primer pair, followed by an NCBI-BLAST comparison. Among these, 32 samples had a 100% match (404/404) with M. speciosa, and 11 samples had a 99.75% match (403/404) with M. speciosa. Among the samples with sequences matching M. speciosa, three distinct types were observed (no variance/404, 287M/404, and 287A/404). One sample had a 99.51% match (404/406) with Neolamarckia cadamba, and another sample had a sequencing length of 305bp, with 25 positions showing mixed base pairs, indicating a mixture of different species. Analysis of the mixed base pair pattern suggested a possible mixture of M. speciosa and N. cadamba. Actually, M. speciosa and N. cadamba have very similar external morphologies. This indicates that the ITS-p3/ITS-u4 primer pair is effective in distinguishing mixtures of M. speciosa and N. cadamba and is thus more suitable than ITS3/ITS4 for identifying and analyzing samples of suspected kratom plant powder.

The sensing of circRNA with tetrahedral DNA nanostructure modified microfluidic chip

BackgroundCircular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. ResultsHerein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. SignificanceThis represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes.

16S rDNA Sequencing for Bacterial Identification in Preterm Infants with Suspected Early-Onset Neonatal Sepsis

Background: The high prevalence of suspected early-onset neonatal sepsis among preterm infants leads to immediate antibiotic administration upon admission. Notably, most blood cultures for suspected early-onset neonatal sepsis do not yield a causative pathogen. This study aimed to assess polymerase chain reaction (PCR) targeting the variable region V4 of the 16S ribosomal gene (16S rDNA) and Sanger sequencing for bacterial identification in preterm infants with suspected early-onset neonatal sepsis. Methods: Therefore, this prospective study was conducted. Preterm infants with suspected early-onset neonatal sepsis were included in this study. The three groups were formed based on the risk of infection and clinical sepsis. Blood samples were collected upon admission to the neonatal unit for culture and molecular analysis. PCR amplification and subsequent Sanger sequencing of the V4 region of the 16S rDNA were performed. Results: Twenty-eight patients were included in this study. Blood cultures were negative in 100% of the patients. Amplification and sequencing of the V4 region identified bacterial genera in 19 patients across distinct groups. The predominant taxonomically identified genus was Pseudomonas. Conclusions: Amplifying the 16S rDNA variable region through PCR and subsequent Sanger sequencing in preterm neonates with suspected early-onset neonatal sepsis can enhance the identification of microbial species that cause infection, especially in negative cultures.

16S rDNA Sequencing for Bacterial Identification in Preterm Infants with Suspected Early-Onset Neonatal Sepsis

Background: The high prevalence of suspected early-onset neonatal sepsis among preterm infants leads to immediate antibiotic administration upon admission. Notably, most blood cultures for suspected early-onset neonatal sepsis do not yield a causative pathogen. This study aimed to assess polymerase chain reaction (PCR) targeting the variable region V4 of the 16S ribosomal gene (16S rDNA) and Sanger sequencing for bacterial identification in preterm infants with suspected early-onset neonatal sepsis. Methods: Therefore, this prospective study was conducted. Preterm infants with suspected early-onset neonatal sepsis were included in this study. The three groups were formed based on the risk of infection and clinical sepsis. Blood samples were collected upon admission to the neonatal unit for culture and molecular analysis. PCR amplification and subsequent Sanger sequencing of the V4 region of the 16S rDNA were performed. Results: Twenty-eight patients were included in this study. Blood cultures were negative in 100% of the patients. Amplification and sequencing of the V4 region identified bacterial genera in 19 patients across distinct groups. The predominant taxonomically identified genus was Pseudomonas. Conclusions: Amplifying the 16S rDNA variable region through PCR and subsequent Sanger sequencing in preterm neonates with suspected early-onset neonatal sepsis can enhance the identification of microbial species that cause infection, especially in negative cultures.

Fatal Hymenolepis nana-associated visceral larva migrans in captive juvenile white-tailed antsangies (Brachytarsomysalbicauda)

White-tailed antsangies (Brachytarsomys albicauda) are Madagascan rodents uncommonly kept in captivity. Hymenolepis nana is a cestode with an unusual life cycle, incorporating direct, indirect and autoinfective stages. This case series represents the first reported outbreak of H. nana cestodiasis in white-tailed antsangies, summarizing macroscopic and histological findings in four cases. On post-mortem examination (PME), numerous cysticerci were detected consistently throughout the intestinal serosa, liver, mesenteric lymphatic vasculature and mesenteric lymph nodes. Pancreatic cysticerci were observed in one case. Adult tapeworms, larvae and eggs were found only in the small intestine, and faecal egg shedding was a feature. Histopathological examination identified adult, larval and encysted cestodes within the respective gross lesions, with pulmonary, pancreatic and splenic involvement detected in a single case. The cestodes sampled on PME were identified by polymerase chain reaction and DNA sequencing, with H. nana confirmed in all cases. Visceral larva migrans was consistent throughout all specimens, in contrast with the natural infections of standard rodent hosts, and may be considered a likely pathological feature of H. nana infection in white-tailed antsangies.

Parotid Gland Tumors: Molecular Diagnostic Approaches.

Parotid gland pathology represents a web of differential diagnoses. There are many complex cases that require extensive diagnostic tests for a complete and correct final pathology diagnosis. Currently the official classification of parotid gland tumors extends over more than 40 subtypes. We performed a query of the PubMed database regarding the use of molecular biology tests in performing a better characterization of the tumors in specific cases. By using fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) or next-generation sequencing, the team managing complex cases can offer a personalized therapeutic solution. We review the molecular differential diagnosis according to published articles in the last 5 years for many types of parotid gland tumors ranging from benign to borderline malign tumors to malign aggressive tumors. Mucoepidermoid carcinoma is a distinct subtype of parotid malignancy that was the subject of a consistent number of articles. However, the molecular biology diagnosis techniques helped more in excluding the diagnosis of mucoepidermoid carcinoma, and probably retrospectively limiting the number of cases with this final diagnosis. In Romania, the molecular biology diagnosis is available only in limited research facilities and should receive more consistent funding that will make it available on a larger scale. The novelty of this scoping review is that we propose an algorithm for molecular differential diagnosis of the tumors that could be encountered in the parotid gland.

Parotid Gland Tumors: Molecular Diagnostic Approaches

Parotid gland pathology represents a web of differential diagnoses. There are many complex cases that require extensive diagnostic tests for a complete and correct final pathology diagnosis. Currently the official classification of parotid gland tumors extends over more than 40 subtypes. We performed a query of the PubMed database regarding the use of molecular biology tests in performing a better characterization of the tumors in specific cases. By using fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) or next-generation sequencing, the team managing complex cases can offer a personalized therapeutic solution. We review the molecular differential diagnosis according to published articles in the last 5 years for many types of parotid gland tumors ranging from benign to borderline malign tumors to malign aggressive tumors. Mucoepidermoid carcinoma is a distinct subtype of parotid malignancy that was the subject of a consistent number of articles. However, the molecular biology diagnosis techniques helped more in excluding the diagnosis of mucoepidermoid carcinoma, and probably retrospectively limiting the number of cases with this final diagnosis. In Romania, the molecular biology diagnosis is available only in limited research facilities and should receive more consistent funding that will make it available on a larger scale. The novelty of this scoping review is that we propose an algorithm for molecular differential diagnosis of the tumors that could be encountered in the parotid gland.

Endothelial Autophagy Promotes Atheroprotective Communication Between Endothelial and Smooth Muscle Cells via Exosome-Mediated Delivery of miR-204-5p.

Cellular communication among different types of vascular cells is indispensable for maintaining vascular homeostasis and preventing atherosclerosis. However, the biological mechanism involved in cellular communication among these cells and whether this biological mechanism can be used to treat atherosclerosis remain unknown. We hypothesized that endothelial autophagy mediates the cellular communication in vascular tissue through exosome-mediated delivery of atherosclerosis-related genes. Rapamycin and adeno-associated virus carrying Atg7 short hairpin RNA under the Tie (TEK receptor tyrosine kinase) promoter were used to activate and inhibit vascular endothelial autophagy in high-fat diet-fed ApoE-/- mice, respectively. miRNA microarray, in vivo and in vitro experiments, and human vascular tissue were used to explore the effects of endothelial autophagy on endothelial function and atherosclerosis and its molecular mechanisms. Quantitative polymerase chain reaction and miRNA sequencing were performed to determine changes in miRNA expression in exosomes. Immunofluorescence and exosome coculture experiments were conducted to examine the role of endothelial autophagy in regulating the communication between endothelial cells and smooth muscle cells (SMCs) via exosomal miRNA. Endothelial autophagy was inhibited in thoracic aortas of high-fat diet-fed ApoE-/- mice. Furthermore, rapamycin alleviated high-fat diet-induced atherosclerotic burden and endothelial dysfunction, while endothelial-specific Atg7 depletion aggravated the atherosclerotic burden. miRNA microarray, in vivo and in vitro experiments, and human vascular tissue analysis revealed that miR-204-5p was significantly increased in endothelial cells after high-fat diet exposure, which directly targeted Bcl2 to regulate endothelial cell apoptosis. Importantly, endothelial autophagy activation decreased excess miR-204-5p by loading miR-204-5p into multivesicular bodies and secreting it through exosomes. Moreover, exosomal miR-204-5p can effectively transport to SMCs, alleviating SMC calcification by regulating target proteins such as RUNX2. Our study revealed the exosomal pathway by which endothelial autophagy protects atherosclerosis: endothelial autophagy activation transfers miR-204-5p from endothelial cells to SMCs via exosomes, both preventing endothelial apoptosis and alleviating SMC calcification. URL: https://www.chictr.org.cn/; Unique identifier: ChiCTR2200064155.

Engineering of a DNA/γPNA Hybrid Nanoreporter for ctDNA Mutation Detection via γPNA Urinalysis.

Detection of circulating tumor DNA (ctDNA) mutations, which are molecular biomarkers present in bodily fluids of cancer patients, can be applied for tumor diagnosis and prognosis monitoring. However, current profiling of ctDNA mutations relies primarily on polymerase chain reaction (PCR) and DNA sequencing and these techniques require preanalytical processing of blood samples, which are time-consuming, expensive, and tedious procedures that increase the risk of sample contamination. To overcome these limitations, here the engineering of a DNA/γPNA (gamma peptide nucleic acid)hybrid nanoreporter is disclosed for ctDNA biosensing via in situ profiling and recording of tumor-specific DNA mutations. The low tolerance of γPNA to single mismatch in base pairing with DNA allows highly selective recognition and recording of ctDNA mutations in peripheral blood. Owing to their remarkable biostability, the detached γPNA strands triggered by mutant ctDNA will be enriched in kidneys and cleared into urine for urinalysis. It is demonstrated that the nanoreporter has high specificity for ctDNA mutation in peripheral blood, and urinalysis of cleared γPNA can provide valuable information for tumor progression and prognosis evaluation. This work demonstrates the potential of the nanoreporter for urinary monitoring of tumor and patient prognosis through in situ biosensing of ctDNA mutations.

Skin microbiome profiling reveals the crucial role of microbial metabolites in anti-photoaging.

Skin microbiota is essential for health maintenance. Photoaging is the primary environmental factor that affects skin homeostasis, but whether it influences the skin microbiota remains unclear. The objective of this study is to investigate the relationship between photoaging and skin microbiome. A cohort of senior bus drivers was considered as a long-term unilateral ultraviolet (UV) irradiated population. 16S rRNA amplicon sequencing was conducted to assess skin microbial composition variations on different sides of their faces. The microbiome characteristics of the photoaged population were further examined by photoaging guinea pig models, and the correlations between microbial metabolites and aging-related cytokines were analyzed by high-throughput sequencing and reverse transcription polymerase chain reaction. Photoaging decreased the relative abundance of microorganisms including Georgenia and Thermobifida in human skin and downregulated the generation of skin microbe-derived antioxidative metabolites such as ectoin. In animal models, Lactobacillus and Streptobacillus abundance in both the epidermis and dermis dropped after UV irradiation, resulting in low levels of skin antioxidative molecules and leading to elevated expressions of the collagen degradation factors matrix metalloproteinase (MMP)-1 and MMP-2 and inflammatory factors such as interleukin (IL)-1β and IL-6. Skin microbial characteristics have an impact in photoaging and the loss of microbe-derived antioxidative metabolites impairs skin cells and accelerates the aging process. Therefore, microbiome-based therapeutics may have potential in delaying skin aging.

Biological and Molecular Characterization of a Korean Isolate of Clover Yellow Vein Virus Infecting Canavalia ensiformis

Jack bean (Canavalia ensiformis) is one of healthy products for fermented or functional food in Korea and is widely distributed and cultivated worldwide. During August 2022, Jack bean plants showing symptoms of yellow flecks, chlorosis, necrotic spots and mosaic were observed in Jangheung-gun, South Korea. By transmission electron microscopy, flexuous filamentous virus particles of approximately 750×13 nm in size were observed in the symptomatic leaf samples. The infection of a Korean isolate of clover yellow vein virus (ClYVV-Ce-JH) was confirmed using double antibody sandwich enzyme-linked sorbent assay, reverse transcription polymerase chain reaction and high-throughput sequencing. The complete genome sequence of ClYVV-Ce-JH consists of 9,549 nucleotides (nt) excluding the poly (A) tail and encodes 3,072 amino acids (aa), with an AUG start and UAG stop codon, containing one open reading frame that is typical of a potyvirus polyprotein. The polyprotein of ClYVV-Ce-JH was divided into ten proteins and each protein’s cleavage sites were determined. The coat protein (CP) and polyprotein of ClYVV-Ce-JH were compared at the nt and aa levels with those of the previously reported 14 ClYVV isolates. ClYVV-Ce-JH shared 92.62% to 99.63% and 93.39% to 98.05% at the CP and polyprotein homology. To our knowledge, this is the first report of a Korean isolate of ClYVV from Jack bean plants and the complete genome sequence of a ClYVV Jack bean isolate in the world.

Archaeal communities in natural and artificially restored mangrove sediments in Tieshan Bay, China

Mangrove forests are crucial wetland ecosystems located in tropical and subtropical intertidal zones, but they have become extensively degraded. As a viable ecological restoration strategy, the cultivation of native mangrove vegetation in these degraded areas has gained considerable attention. Mangroves’ unique environmental conditions make them suitable habitats for diverse microbial communities, including Archaea - one of the main microbial communities in mangrove sediments - which plays a pivotal role in biogeochemical cycles. However, little is known about the dynamics of archaeal communities during mangrove restoration through phytoremediation. In this study, we investigated the physicochemical properties of sediment profiles from natural and artificially restored mangrove ecosystems in Tieshan Bay. We utilized quantitative polymerase chain reaction (qPCR) and 16S rRNA gene sequencing techniques to explore differences in abundance, community structure, and composition of archaeal communities between sediment profiles in natural and artificially restored mangrove ecosystems. We also examined correlations between archaeal communities and environmental factors. Our results revealed that Crenarchaeota, Thermoplasmatota, Asgardarchaeota, Nanoarchaeota, and Euryarchaeota were the predominant archaeal phyla, with significant variation in sediment composition observed for Crenarchaeota and Thermoplasmatota in different depths. We also found significant differences in archaeal abundance and community composition between natural and restored mangrove sediments. Furthermore, C/N ratio and pH emerged as primary drivers of archaeal communities in wet and dry season sediments, respectively. Additionally, the study revealed seasonal disparities in seasonal differences in the relative abundance of certain archaeal groups (such as Asgardarchaeota, Nanoarchaeota). Network analysis demonstrated stronger interconnections among archaeal communities in sediments from natural mangroves than from artificially restored ones. These findings enhance our knowledge of archaeal community succession patterns in mangrove restoration, as well as provide fresh perspectives for the sustainable management of mangrove ecosystems.

The diversity and risk of potential pathogenic bacteria on the surface of glaciers in the southeastern Tibetan Plateau

Glaciers, which constitute the world's largest global freshwater reservoir, are also natural microbial repositories. The frequent pandemic in recent years underscored the potential biosafety risks associated with the release of microorganisms from the accelerated melting of glaciers due to global warming. However, the characteristics of pathogenic microorganisms in glaciers are not well understood. The glacier surface is the primary area where glacier melting occurs that is often the main subject of research on the dynamics of pathogenic microbial communities in efforts to assess glacier biosafety risks and devise preventive measures. In this study, high-throughput sequencing and quantitative polymerase chain reaction methods were employed in analyses of the composition and quantities of potential pathogenic bacteria on the surfaces of glaciers in the southeastern Tibetan Plateau. The study identified 441 potential pathogenic species ranging from 215 to 4.39 × 1011 copies/g, with notable seasonal and environmental variations being found in the composition and quantity of potential pathogens. The highest level of diversity was observed in April and snow, while the highest quantities were observed in October and cryoconite. Host analysis revealed that >70 % of the species were pathogens affecting animals, with the highest proportion of zoonotic pathogens being observed in April. Analysis of aerosols and glacial meltwater dispersion suggested that these microbes originated from West Asia, primarily affecting the central and southern regions of China. Null model analysis indicated that the assembly of potential pathogenic microbial communities on glacier surfaces was largely governed by deterministic processes. In conclusion, potential pathogenic bacteria on glacier surfaces mainly originated from the snow and exhibited significant temporal and spatial variation patterns. These findings can be used to enhance researchers' ability to predict potential biosafety risks associated with pathogenic bacteria in glaciers and to prevent their negative impact on populations and ecological systems.

Evaluation of diagnostic techniques for early detection of heartworm in experimentally infected dogs: identification of Dirofilaria immitis-derived microRNA in the initial 28 weeks post-inoculation

BackgroundDirofilaria immitis, commonly known as heartworm (HW), is a parasitic nematode transmitted by various mosquito species, leading to heartworm disease (HWD) in dogs. Diagnosis of HW typically involves antigen or microfilariae detection, or visualization of adult worms through imaging or post mortem examination. Polymerase chain reaction (PCR) and micro RNA (miRNA) detection have been explored for HW diagnosis.MethodsThree dogs, previously experimentally infected with HW, underwent blood sampling every 4 weeks for 7 months. Samples were assessed for antigen presence after heat treatment, PCR amplification, and microfilaria examination using Giemsa-stained thick smears. Additionally, whole blood aliquots underwent miRNA deep sequencing and bioinformatic analysis.ResultsHeartworm antigen was detectable after heat treatment at 20 weeks post-inoculation and via PCR at 24 weeks, with microfilariae observed in peripheral blood smears at 28 weeks. However, deep miRNA sequencing revealed that the miRNA candidate sequences are not consistently expressed before 28 weeks of infection.ConclusionsWhile ancillary molecular methods such as PCR and miRNA sequencing may be less effective than antigen detection for detecting immature larval stages in an early stage of infection, our experimental findings demonstrate that circulating miRNAs can still be detected in 28 weeks post-infection.Graphical

TRIM35 Monoubiquitinates H2B in Cardiac Cells, Implications for Heart Failure.

The tumor suppressor and proapoptotic transcription factor P53 is induced (and activated) in several forms of heart failure, including cardiotoxicity and dilated cardiomyopathy; however, the precise mechanism that coordinates its induction with accessibility to its transcriptional promoter sites remains unresolved, especially in the setting of mature terminally differentiated (nonreplicative) cardiomyocytes. Male and female control or TRIM35 (tripartite motif containing 35) overexpression adolescent (aged 1-3 months) and adult (aged 4-6 months) transgenic mice were used for all in vivo experiments. Primary adolescent or adult mouse cardiomyocytes were isolated from control or TRIM35 overexpression transgenic mice for all in vitro experiments. Adenovirus or small-interfering RNA was used for all molecular experiments to overexpress or knockdown, respectively, target genes in primary mouse cardiomyocytes. Patient dilated cardiomyopathy or nonfailing left ventricle samples were used for translational and mechanistic insight. Chromatin immunoprecipitation and DNA sequencing or quantitative real-time polymerase chain reaction (qPCR) was used to assess P53 binding to its transcriptional promoter targets, and RNA sequencing was used to identify disease-specific signaling pathways. Here, we show that E3-ubiquitin ligase TRIM35 can directly monoubiquitinate lysine-120 (K120) on histone 2B in postnatal mature cardiomyocytes. This epigenetic modification was sufficient to promote chromatin remodeling, accessibility of P53 to its transcriptional promoter targets, and elongation of its transcribed mRNA. We found that increased P53 transcriptional activity (in cardiomyocyte-specific Trim35 overexpression transgenic mice) was sufficient to initiate heart failure and these molecular findings were recapitulated in nonischemic human LV dilated cardiomyopathy samples. These findings suggest that TRIM35 and the K120Ub-histone 2B epigenetic modification are molecular features of cardiomyocytes that can collectively predict dilated cardiomyopathy pathogenesis.

Transcriptome sequencing and bioinformatics analysis of gastrocnemius muscle in type 2 diabetes mellitus rats

ObjectiveType 2 diabetes mellitus (T2DM) is one of the high risk factors for sarcopenia. However, the pathogenesis of diabetic sarcopenia has not been fully elucidated. This study obtained transcriptome profiles of gastrocnemius muscle in normal and T2DM rats based on high-throughput sequencing technology, which may provide new ideas for exploring the pathogenesis of diabetic sarcopenia.MethodsTwelve adult male Sprague-Dawley rats were randomly divided into Control group and T2DM group, and gastrocnemius muscle tissue was retained for transcriptome sequencing and real-time quantitative polymerase chain reaction (qRT-PCR) 6 months later. Screening differentially expressed genes (DEGs), Cluster analysis, gene ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Gnomes (KEGG) functional annotation and enrichment analysis were performed for DEGs. Six DEGs related to apoptosis were selected for qTR-PCR verification.ResultsTranscriptomic analysis showed that there were 1016 DEGs between the gastrocnemius muscle of T2DM and normal rats, among which 665 DEGs were up-regulated and 351 DEGs were down-regulated. GO analysis showed that the extracellular matrix organization was the most enriched in biological processes, with 26 DEGs. The extracellular matrix with 35 DEGs was the most abundant cellular component. The extracellular matrix structural constituent, with 26 DEGs, was the most enriched in molecular functions. The highest number of DEGs enriched in biological processes, cellular components and molecular functions were positive regulation of transcription by RNA polymerase II, nucleus and metal ion binding, respectively. There were 78, 230 and 89 DEGs respectively. KEGG pathway enrichment analysis showed that ECM-receptor interaction, PI3K-Akt signaling pathway and TGF-β signaling pathway(p < 0.001) had higher enrichment degree and number of DEGs. qRT-PCR results showed that the fold change of Map3k14, Atf4, Pik3r1, Il3ra, Gadd45b and Bid were 1.95, 3.25, 2.97, 2.38, 0.43 and 3.6, respectively. The fold change of transcriptome sequencing were 3.45, 2.21, 2.59, 5.39, 0.49 and 2.78, respectively. The transcriptional trends obtained by qRT-PCR were consistent with those obtained by transcriptome sequencing.ConclusionsTranscriptomic analysis was used to obtain the “gene profiles” of gastrocnemius muscle of T2DM and normal rats. qRT-PCR verification showed that the genes related to apoptosis were differentially expressed. These DEGs and enrichment pathways may provide new ideas for exploring the pathogenesis of diabetic sarcopenia.