Increased Gene Copy Number Research Articles

Diversity and Evolution of NLR Genes in Citrus Species.

NLR genes are crucial components of the effector-triggered immunity (ETI) system, responsible for recognizing pathogens and initiating immune responses. Although NLR genes in many plant species have been extensively studied, the diversity of NLR genes in citrus remains largely unknown. Our analysis revealed significant variations in the copy numbers of NLR genes among these species. Gene duplication and recombination were identified as the major driving forces behind this diversity. Additionally, horizontal gene transfer (HGT) emerged as the principal mechanism responsible for the increase in NLR gene copy number in A. buxifolia. The citrus NLR genes were classified into four categories: TIR-NBS-LRR (TNL), CC-NBS-LRR (CNL), RPW8-NBS-LRR (RNL), and NL. Our findings indicate that TNL, RNL, and CNL genes originated from NL genes through the acquisition of TIR and RPW8 domains, along with CC motifs, followed by the random loss of corresponding domains. Phylogenetic analysis suggested that citrus NLR genes originated alongside the species and underwent adaptive evolution, potentially playing crucial roles in the global colonization of citrus. This study provides important insights into the diversity of citrus NLR genes and serves as a foundational dataset for future research aimed at breeding disease-resistant citrus varieties.

Modified Tn7 transposon vectors for controlled chromosomal gene expression.

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing differential transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted.IMPORTANCEChromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology.

CDK4 gene copy number increase and concurrent genetic changes in acral melanoma of a Chinese cohort

Acral melanoma (AM) is the most common subtype of melanoma in the Asian population. Abnormalities in the p16-cyclin D1-CDK4 signalling pathway play a crucial role in the development and progression of AM. However, the CDK4 copy number variations (CNVs) in AM are under-reported. In this study, we investigated CDK4 gene copy number and concurrent molecular changes in a Chinese cohort with AM, to explore CDK4 CNVs and their significance in AM. We examined CDK4 CNVs with fluorescence in situ hybridisation (FISH) in 31 patients with AM. Six patients with CDK4 high-level copy number increase were examined by next-generation sequencing to detect concurrent molecular changes. Using FISH, 12 (12/31, 38.7%) cases showed CDK4 copy number increase, with six (6/31, 19.4%) low-level copy number increase and six (6/31, 19.4%) high-level copy number increase. Five of six CDK4 low-level copy number increase cases were accompanied by polysomy of chromosome 12, while one case was not. Two of six CDK4 high-level copy number increase cases were accompanied by polysomy of chromosome 12, while four cases were not. CDK4 copy number increase was significantly correlated with younger patient age. In six CDK4 high-level copy number increase cases, one case was found to be accompanied by NRAS mutation, one case was accompanied by HER2 mutation, one case was accompanied by BCL2L11 mutation ​and one case was accompanied by BRAF, HER2 and BCL2L11 mutations. Our study confirmed the presence of CDK4 copy number increase in AM cases. Detecting CDK4 copy number increase by FISH can be reliable in the diagnosis of AM. Some CDK4 copy number increases are the results of polysomy of chromosome 12. CDK4 high-level copy number increase coexists with other pathogenic mutations in AM. CDK4 appears to be a promising target for AM treatment and is expected to be combined with other targeted therapies.

Association between the copy number variations of Methyl-CpG binding domain family and schizophrenia

Schizophrenia is recognized as one of the most severe psychiatric disorders, with its pathogenesis likely involving genetic, epigenetic, developmental, and environmental factors. Members of the Methyl-CpG Binding Domain (MBD) Family play a crucial role in the regulation of genomic DNA methylation, and studies have implicated the association between MBD family and neurodevelopmental disorders. Copy number variations (CNVs) are a significant genetic basis for human genomic variation, also playing a critical role in the genetic processes of schizophrenia. Therefore, we aimed to evaluate the susceptibility of MBD family CNVs to schizophrenia by exploring and validating them in two separate populations using CNVplex™ and qPCR methods, and to explore the relationship between MBD family CNVs and clinical phenotypes in the overall population using chi-square tests and Fisher’s exact tests. Results suggest that an increase in MBD1 gene copy number and a deficiency in MBD2 gene copy number may be associated with the risk of schizophrenia. The deficiency in MBD2 gene copy number may increase the risk of delusion of reference and delusion of persecutory in the overall sample, as well as in males. This research provides preliminary evidence supporting the association between MBD family CNVs and schizophrenia, highlighting the potential role of the MBD family in the pathogenesis of schizophrenia.

Field test of a bioaugmentation agent for the bioremediation of chlorinated ethene contaminated sites.

Chlorinated ethenes are toxic compounds that were widely used in the past, and their improper handling and storage caused notable pollutions worldwide. In situ bioremediation by reductive dechlorination of bacteria is a cost-effective and ecologically friendly way to eliminate these pollutions. During the present study, the efficiency of a previously developed bioaugmentation agent combined with biostimulation was tested under field conditions in contaminated soil. Furthermore, the preservation of dechlorinating ability was also investigated in a long-term experiment. Initially, aerobic conditions were present in the groundwater with possible presence of anaerobic micro-niches providing habitat for Brocadia related anammox bacteria. "Candidatus Omnitrophus" was also identified as a dominant member of community then. Significant changes were detected after the biostimulation, anaerobic conditions established and most of the dominant OTUs were related to fermentative taxa (e.g. Clostridium, Trichococcus and Macillibacteroides). Dominant presence of vinyl-chloride coupled with the lack of vinyl-chloride reductase gene was observed. The most notable change after the bioaugmentation was the significant decrease in the pollutant quantities and the parallel increase in the vcrA gene copy numbers. Similar to post-biostimulation state, fermentative bacteria dominated the community. Bacterial community composition transformed considerably with time after the treatment, dominance of fermentative-mainly Firmicutes related-taxa decreased and chemolithotrophic bacteria became abundant, but the dechlorinating potential of the community remained and could be induced by the reappearance of the pollutants even after 4 years.

Duplicate Gene Expression and Possible Mechanisms of Paralog Retention During Bacterial Genome Expansion.

Gene duplication contributes to the evolution of expression and the origin of new genes, but the relative importance of different patterns of duplicate gene expression and mechanisms of retention remains debated and particularly poorly understood in bacteria. Here, we investigated gene expression patterns for two lab strains of the cyanobacterium Acaryochloris marina with expanding genomes that contain about 10-fold more gene duplicates compared with most bacteria. Strikingly, we observed a generally stoichiometric pattern of greater combined duplicate transcript dosage with increased gene copy number, in contrast to the prevalence of expression reduction reported for many eukaryotes. We conclude that increased transcript dosage is likely an important mechanism of initial duplicate retention in these bacteria and may persist over long periods of evolutionary time. However, we also observed that paralog expression can diverge rapidly, including possible functional partitioning, for which different copies were respectively more highly expressed in at least one condition. Divergence may be promoted by the physical separation of most Acaryochloris duplicates on different genetic elements. In addition, expression pattern for ancestrally shared duplicates could differ between strains, emphasizing that duplicate expression fate need not be deterministic. We further observed evidence for context-dependent transcript dosage, where the aggregate expression of duplicates was either greater or lower than their single-copy homolog depending on physiological state. Finally, we illustrate how these different expression patterns of duplicated genes impact Acaryochloris biology for the innovation of a novel light-harvesting apparatus and for the regulation of recA paralogs in response to environmental change.

Regulation of nitrogen transformation and microbial community by inoculation during livestock manure composting.

This study examined the effects of three Bacillus strains and one Saccharomyces cerevisiae strain on nitrogen transformation and microbial communities in pig and chicken manure compost. The findings revealed that the use of compound microbial inoculants increased the compost temperature, accelerated moisture reduction, enhanced cellulase activity, and stimulated the accumulation of NH4 +-N, NO3 --N, and total nitrogen (TN), resulting in a 9% increase in TN content. The abundance of Firmicutes decreased by 3.95% at the maturation phase, while Actinobacteria and Bacteroidetes increased by 1.64% and 1.85%, respectively. Inoculation led to an increase in amoA, nxrA and nifH gene copy numbers, while simultaneously reducing the abundance of nirK, nosZ and nirS genes. It also resulted in an increase in functional enzyme levels, specifically nif and amo, with a corresponding decrease in nor. Clostridium, Phascolarctobacterium, Eubacterium and Faecalibacterium from the class Clostridium, which have a significant correlation with nifH and nxrA genes, suggest their likely crucial role in nitrogen retention and fixation. Inoculation aided in the removal of pathogenic bacteria and antibiotic resistance genes (ARGs) like fluoroquinolones, nucleosides and nitroimidazole. This study provides effective theoretical support for the mechanism of nitrogen retention and fixation, and for improving the quality of compost.

Bacteria can compensate the fitness costs of amplified resistance genes via a bypass mechanism

Antibiotic heteroresistance is a phenotype in which a susceptible bacterial population includes a small subpopulation of cells that are more resistant than the main population. Such resistance can arise by tandem amplification of DNA regions containing resistance genes that in single copy are not sufficient to confer resistance. However, tandem amplifications often carry fitness costs, manifested as reduced growth rates. Here, we investigated if and how these fitness costs can be genetically ameliorated. We evolved four clinical isolates of three bacterial species that show heteroresistance to tobramycin, gentamicin and tetracyclines at increasing antibiotic concentrations above the minimal inhibitory concentration (MIC) of the main susceptible population. This led to a rapid enrichment of resistant cells with up to an 80-fold increase in the resistance gene copy number, an increased MIC, and severely reduced growth rates. When further evolved in the presence of antibiotic, these strains acquired compensatory resistance mutations and showed a reduction in copy number while maintaining high-level resistance. A deterministic model indicated that the loss of amplified units was driven mainly by their fitness costs and that the compensatory mutations did not affect the loss rate of the gene amplifications. Our findings suggest that heteroresistance mediated by copy number changes can facilitate and precede the evolution towards stable resistance.

Alternative Starter Fertilization Strategies in Maize (Zea mays L.) Cultivation: Agronomic Potential of Microgranular Fertilizer and Plant Growth-Promoting Microorganisms and Their Impact on the Soil Native Microbial Community

Phosphorous (P) starter fertilization can increase maize (Zea mays L.) yield. Widespread application in soils with sufficient P availability leads to environmental risks. Subsequently, alternative strategies to support the maize plant’s early development are needed to lower P surpluses. Here, we conducted field experiments comparing standard starter fertilizer diammonium phosphate (DAP) (20.1 kg P ha−1) to microgranular fertilizer (MG) (2.4 kg P ha−1) and combined in-furrow inoculation with Bacillus atrophaeus and mycorrhizal fungi (Rhizoglomus irregulare, Funneliformis mosseae, and Funneliformis caledonium), alone and in combination. The soil microbial community inside and between the maize rows was monitored by quantitative PCR (qPCR)-based quantification of eight fungal and bacterial groups. The yield did not vary between fertilization with DAP or MG and no fertilizer control. The combined microorganism inoculum (MO), however, enhanced the yield by 4.2%. The soil microbial community composition was not affected by the MO application. However, on one field site and inside the rows, it leads to a significant increase in overall microbial gene copy numbers by 9.3% and a significant decrease in the relative abundance of the bacterial phylum of Bacillota (Firmicutes) by 18%. The in-furrow MO application is thus a promising option for starter fertilizer replacement.

Genomics of predictive radiation mutagenesis in oilseed rape: modifying seed oil composition.

Rapeseed is a crop of global importance but there is a need to broaden the genetic diversity available to address breeding objectives. Radiation mutagenesis, supported by genomics, has the potential to supersede genome editing for both gene knockout and copy number increase, but detailed knowledge of the molecular outcomes of radiation treatment is lacking. To address this, we produced a genome re-sequenced panel of 1133 M2 generation rapeseed plants and analysed large-scale deletions, single nucleotide variants and small insertion-deletion variants affecting gene open reading frames. We show that high radiation doses (2000 Gy) are tolerated, gamma radiation and fast neutron radiation have similar impacts and that segments deleted from the genomes of some plants are inherited as additional copies by their siblings, enabling gene dosage decrease. Of relevance for species with larger genomes, we showed that these large-scale impacts can also be detected using transcriptome re-sequencing. To test the utility of the approach for predictive alteration of oil fatty acid composition, we produced lines with both decreased and increased copy numbers of Bna.FAE1 and confirmed the anticipated impacts on erucic acid content. We detected and tested a 21-base deletion expected to abolish function of Bna.FAD2.A5, for which we confirmed the predicted reduction in seed oil polyunsaturated fatty acid content. Our improved understanding of the molecular effects of radiation mutagenesis will underpin genomics-led approaches to more efficient introduction of novel genetic variation into the breeding of this crop and provides an exemplar for the predictive improvement of other crops.

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate‑resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double‑strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)‑resistant HT29 cells [DM‑ and homogeneously staining region (HSR)‑containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM‑containing cells. No corresponding changes were found in HSR‑containing cells. The present study further verified the involvement of MSH3‑regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3‑depleted DM‑containing cells. Finally, changes in MSH3 expression could affect DHFR amplification‑derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA‑based drug resistance in cancer.

The genome of Anoplarchus purpurescens (Stichaeidae) reflects its carnivorous diet

Digestion is driven by digestive enzymes and digestive enzyme gene copy number can provide insights on the genomic underpinnings of dietary specialization. The “Adaptive Modulation Hypothesis” (AMH) proposes that digestive enzyme activity, which increases with increased gene copy number, should correlate with substrate quantity in the diet. To test the AMH and reveal some of the genetics of herbivory vs carnivory, we sequenced, assembled, and annotated the genome of Anoplarchus purpurescens, a carnivorous prickleback fish in the family Stichaeidae, and compared the gene copy number for key digestive enzymes to that of Cebidichthys violaceus, a herbivorous fish from the same family. A highly contiguous genome assembly of high quality (N50 = 10.6 Mb) was produced for A. purpurescens, using combined long-read and short-read technology, with an estimated 33,842 protein-coding genes. The digestive enzymes that we examined include pancreatic α-amylase, carboxyl ester lipase, alanyl aminopeptidase, trypsin, and chymotrypsin. Anoplarchus purpurescens had fewer copies of pancreatic α-amylase (carbohydrate digestion) than C. violaceus (1 vs. 3 copies). Moreover, A. purpurescens had one fewer copy of carboxyl ester lipase (plant lipid digestion) than C. violaceus (4 vs. 5). We observed an expansion in copy number for several protein digestion genes in A. purpurescens compared to C. violaceus, including trypsin (5 vs. 3) and total aminopeptidases (6 vs. 5). Collectively, these genomic differences coincide with measured digestive enzyme activities (phenotypes) in the two species and they support the AMH. Moreover, this genomic resource is now available to better understand fish biology and dietary specialization.

Sub-micron microplastics affect nitrogen cycling by altering microbial abundance and activities in a soil-legume system

Recently, the environmental and agricultural impact of plastic waste has attracted considerable attention. Here, we investigated the impact of sub-micron polyethylene (PE) and polypropylene (PP) microplastics (MPs) on nitrogen cycling, with emphasis on bacterial abundance and diversity in a soil-soybean (Glycine max) system. Exposure to soil containing MPs (50 and 500 mg kg−1) did not affect soybean growth, but significantly increased plant nitrogen uptake, which was confirmed by increased activities of nitrogenase in the soil and glutamine synthetase in soybean root. Additionally, there was an increase in 16S gene copy number and carbon and nitrogen substrate utilization, indicating increased abundance and activity of rhizosphere microbial communities. Moreover, MP contamination affected the taxonomic profile of rhizosphere bacteria, especially the abundance of symbiotic and free-living bacteria involved in nitrogen cycling. Furthermore, qPCR analysis of nitrogen-related genes and Kyoto Encyclopedia of Genes and Genomes analysis of 16S rRNA gene sequencing data revealed an increased abundance of functional genes associated with nitrogen fixation and nitrification. However, the concentration and polymer type of MPs did not have a significant impact in our system. Overall, these results provide insights into the interactions between MPs and rhizosphere bacterial communities in the soil-legume system.

Host-diet-gut microbiome interactions influence human energy balance: a randomized clinical trial

The gut microbiome is emerging as a key modulator of human energy balance. Prior studies in humans lacked the environmental and dietary controls and precision required to quantitatively evaluate the contributions of the gut microbiome. Using a Microbiome Enhancer Diet (MBD) designed to deliver more dietary substrates to the colon and therefore modulate the gut microbiome, we quantified microbial and host contributions to human energy balance in a controlled feeding study with a randomized crossover design in young, healthy, weight stable males and females (NCT02939703). In a metabolic ward where the environment was strictly controlled, we measured energy intake, energy expenditure, and energy output (fecal and urinary). The primary endpoint was the within-participant difference in host metabolizable energy between experimental conditions [Control, Western Diet (WD) vs. MBD]. The secondary endpoints were enteroendocrine hormones, hunger/satiety, and food intake. Here we show that, compared to the WD, the MBD leads to an additional 116 ± 56 kcals (P < 0.0001) lost in feces daily and thus, lower metabolizable energy for the host (89.5 ± 0.73%; range 84.2-96.1% on the MBD vs. 95.4 ± 0.21%; range 94.1-97.0% on the WD; P < 0.0001) without changes in energy expenditure, hunger/satiety or food intake (P > 0.05). Microbial 16S rRNA gene copy number (a surrogate of biomass) increases (P < 0.0001), beta-diversity changes (whole genome shotgun sequencing; P = 0.02), and fermentation products increase (P < 0.01) on an MBD as compared to a WD along with significant changes in the host enteroendocrine system (P < 0.0001). The substantial interindividual variability in metabolizable energy on the MBD is explained in part by fecal SCFAs and biomass. Our results reveal the complex host-diet-microbiome interplay that modulates energy balance.

Grapevine genome analysis demonstrates the role of gene copy number variation in the formation of monoterpenes.

Volatile organic compounds such as terpenes influence the quality parameters of grapevine through their contribution to the flavour and aroma profile of berries. Biosynthesis of volatile organic compounds in grapevine is relatively complex and controlled by multiple genes, the majority of which are unknown or uncharacterised. To identify the genomic regions that associate with modulation of these compounds in grapevine berries, volatile metabolic data generated via GC-MS from a grapevine mapping population was used to identify quantitative trait loci (QTLs). Several significant QTLs were associated with terpenes, and candidate genes were proposed for sesquiterpene and monoterpene biosynthesis. For monoterpenes, loci on chromosomes 12 and 13 were shown to be associated with geraniol and cyclic monoterpene accumulation, respectively. The locus on chromosome 12 was shown to contain a geraniol synthase gene (VvGer), while the locus on chromosome 13 contained an α-terpineol synthase gene (VvTer). Molecular and genomic investigation of VvGer and VvTer revealed that these genes were found in tandemly duplicated clusters, displaying high levels of hemizygosity. Gene copy number analysis further showed that not only did VvTer and VvGer copy numbers vary within the mapping population, but also across recently sequenced Vitis cultivars. Significantly, VvTer copy number correlated with both VvTer gene expression and cyclic monoterpene accumulation in the mapping population. A hypothesis for a hyper-functional VvTer allele linked to increased gene copy number in the mapping population is presented and can potentially lead to selection of cultivars with modulated terpene profiles. The study highlights the impact of VvTPS gene duplication and copy number variation on terpene accumulation in grapevine.

P16 and EGFR analyses in cervical carcinoma on paraffin material in Egyptian females: the NCI experience

Abstract Introduction Cervical cancer is considered the fourth most prevalent malignant tumor in women worldwide. In Egypt, it ranks as the 14th most frequent cancer among women. Epidermal growth factor receptor has been an attractive target for anticancer therapy. Although EGFR mutations are less commonly seen in cervical cancer, there is a potential correlation between EGFR expression level and clinical response. Overexpression of p16INK4a is associated with increased overall and disease-free survival in cervical cancer, thus denoting a better prognosis. Aim of the work To investigate the expression of EGFR and p16 by immunohistochemistry as well as amplification of EGFR by FISH technique in cervical carcinoma among Egyptian females. Material and methods Seventy-seven cases of invasive cervical carcinomas were retrieved from paraffin blocks. EGFR and p16 expression were assessed by immunohistochemistry. EGFR gene amplification was assessed by FISH analysis. Results p 16 Positivity was detected in 62.3% of cases. Positive reaction to EGFR (score 3+) was detected in 20.8% of cases. The EGFR gene was not amplified in 77.9% of cases. Statistical significance was found between histological type, p16, and EGFR expression. A fair agreement was detected between EGFR expression and EGFR amplification. Poor agreement was detected between p16, EGFR expression, and EGFR amplification. Conclusion EGFR and p16 have an independent role in the development of cervical cancer in Egyptian females. EGFR overexpression and EGFR amplification represent two different genetic events; in other words, an increase in gene copy number does not mean that there will be an increase in the function of the gene as expressed by an increase in protein expression.

Q-PCR Methodology for Monitoring the Thermophilic Hydrogen Producers Enriched from Elephant Dung

This study aims to create a quantitative polymerase chain reaction (q-PCR) methodology for monitoring the hydrogen-producing mixed cultures enriched from elephant dung using alpha-cellulose as a carbon source through five generations of repetitive sub-culture. The enriched thermophilic mixed cultures from the fifth cultivation cycle gave the highest hydrogen yield of 170.3 mL H2/g cellulose and were used to generate hydrogen from sawdust. Clostridium sp. and Thermoanaerobacterium sp. were the dominant bacteria in thermophilic mixed cultures with high hydrogen yield, according to polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE). q-PCR primers Chis150F and ClostIR, TherF and TherR, and BacdF and BacdR were developed to amplify the 16S rRNA genes of Clostridium sp., Thermoanaerobacterium sp., and Bacillus sp., respectively, for the quantification of hydrogen-producing bacteria in biohydrogen fermentation. Similar q-PCR analysis of Clostridium sp., Thermoanaerobacterium sp., and Bacillus sp. 16S rRNA gene amplification during hydrogen production from cellulose and sawdust revealed increasing gene copy number with time. The molecular approaches developed in this study can be used to monitor microbial communities in hydrogen fermentation processes efficiently.

Hydrochar amendments stimulate soil nitrous oxide emission by increasing production of hydroxyl radicals and shifting nitrogen functional genesin the short term: A culture experiment.

The application of waste biomass-derived hydrochar to soil may cause extremely intensive nitrous oxide (N2O) fluxes that can challenge our current mechanistic understanding of the global nitrogen cycle in the biosphere. In this study, two waste biomasses were used to prepare cyanobacterial biomas-derived hydrochar (CHC) and wheat straw-derived hydrochar (SHC) for short-term incubation experiments to identify their effects and mechanisms of waste biomass-derived hydrochar on soil N2O efflux, with time-series samples collected for N2O efflux and soil analysis. The results showed that CHC and SHC caused short-term bursts of N2O effluxes without nitrogen inputs. Moreover, the enrichment of exogenous organics and nutrients at the hydrochar-soil interface was identified as the key factor for enhancing N2O fluxes, which stimulated microbial nitrification (i.e., increased gene copy number of ammonia oxidizing bacteria) and denitrification (i.e., increased gene copy number of nitrate and N2O reducing bacteria) processes. The concentrations of Fe (II) and hydroxyl radicals (HO•) were 6.49 and 5.63 times higher, respectively, in the hydrochar layer of CHC than SHC amendment. Furthermore, structural equation models demonstrated that HO•, as well as soil microbiomes, played an important role in driving N2O fluxes. Together, our findings provide a deeper insight into the assessment and prognosis of the short-term environmental risk arising from agricultural waste management in integrated agriculture. Further studies under practical field application conditions are warranted to verify the findings.

Engineering heterologous enzyme secretion in Yarrowia lipolytica

BackgroundEukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme.ResultsBy engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and alpha-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion.ConclusionsOverall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.

Angiofibroma of Soft Tissue: A Clinicopathological Study of Eight Cases With Emphasis on the Diagnostic Utility of Fluorescence In Situ Hybridization Detection for NCOA2 Rearrangement.

BackgroundAngiofibroma of soft tissue (AFST) is a rare mesenchymal neoplasm of fibroblastic differentiation. Due to its diverse morphology and the lack of specific immunohistochemistry (IHC) markers, AFST could elicit a broad range of differential diagnosis. Several studies have disclosed in AFST recurrent gene fusions involving NCOA2, mainly AHRR–NCOA2 fusion, providing a useful approach to diagnosing this lesion. We report eight additional cases of this rare entity with emphasis on the diagnostic utility of fluorescence in situ hybridization (FISH) detection for NCOA2 rearrangement.MethodsClinicopathological data for eight AFSTs were retrieved. IHC was performed, and FISH was used to detect rearrangements involving NCOA2, DDIT3, and FUS loci.ResultsThere were five female and three male patients, ranging in age from 29 to 69 years (median: 55 years). The patients presented mostly with a slow-growing mass in the extremities, with or without intermittent pain. All tumors were located in the lower extremities with three (27.5%) involving or adjacent to the knee joints. Tumor size ranged from 1.5 to 3.8 cm (median: 3.0 cm). Morphologically, the tumors consisted of a proliferation of uniform, bland spindle cells set in alternating myxoid and collagenous stroma with a prominent vascular network composed of countless small, branching, thin-walled blood vessels. Foci of “chicken wire”-like capillaries and medium- to large-sized blood vessels with prominent staghorn morphology were evident in two and four cases, respectively. In addition, sheets of small round cells and foci of cystic changes were observed in one each case. Degenerative nuclear atypia was identified in three cases, while mitosis and tumor necrosis were absent. By IHC, the stromal cells were variably positive for epithelial membrane antigen, desmin, and CD68. By FISH analysis, seven out of eight cases (87.5%) showed NCOA2 rearrangement, and the remaining one had increased gene copy numbers of intact NCOA2; rearrangements involving FUS (0/4) and DDIT3 (0/3) were not identified in the cases analyzed. All tumors were surgically removed, and none had recurrence at follow-up from 5 to 73 months.ConclusionsFISH analysis for NCOA2 rearrangement represents a practical method for confirming the diagnosis of AFST on the basis of appropriate histomorphological backgrounds.