Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics.

As human progenitor cells differentiate into neurons, the activities of many genes change; these changes are maintained within a narrow range, referred to as genome homeostasis. This process, which alters the synchronization of the entire expressed genome, is distorted in neurodevelopmental diseases such as schizophrenia. The coordinated gene activity networks formed by altering sets of genes comprise recurring coordination modules, governed by the entropy-controlling action of nuclear FGFR1, known to be associated with DNA topology. These modules can be modeled as energy-transferring circuits, revealing that genome homeostasis is maintained by reducing oscillations (noise) in gene activity while allowing gene activity changes to be transmitted across networks; this occurs more readily in neuronal committed cells than in neural progenitors. These findings advance a model of an \"entangled\" global genome acting as a flexible, coordinated homeostatic system that responds to developmental signals, is governed by nuclear FGFR1, and is reprogrammed in disease.

D-Pantothenic acid, as a momentous vitamin, is extensively applied to feed, medicine, cosmetics and other fields. However, there are still limitations to produce D-pantothenic acid by microbial fermentation at present. In this paper, we constructed a recombinant strain for D-pantothenic acid production by blocking the organic acid pathway, boosting pyruvate biosynthesis, relieving feedback inhibition of acetolactate synthase, improving glucose intake capacity, and modifying essential genes in the metabolic pathway. In addition, a new acetolactate isomeroreductase mutant V412A origin from Escherichia coli (EcAHAIR) encoded by ilvC was obtained to explore its substrate promiscuity. Compared with the wild type, the variant EcAHAIR-V412A has reduced steric hindrance and enhanced intermolecular forces, resulting in a high affinity for 2-acetolactate. Eventually, the fermentation production of the final strain DPAN19/trc-ilvCV412A reached 4.65 g/L, increased by 192.5% compared with strain DPA8 in shake flask cultivation and produced 62.82 g/L D-pantothenic acid in a 5 L bioreactor. The metabolic engineering strategies and enzyme modification approaches described in this paper provide a particular perspective for the bio-manufacturing of D-pantothenic acid, branched-chain amino acids and its derivates.

Ergothioneine (EGT) is a rare thiohistidine derivative with exceptional antioxidant properties. The blood level of EGT is considered highly reliable predictors for cardiovascular diseases and mortality, yet animals lack the ability to synthesize this compound. Free plasmids have been previously used to overexpress genes involved in the EGT biosynthetic pathway of Mycolicibacterium neoaurum. Here, we tentatively introduced a putative transporter gene mfsT1 into high-copy plasmids and sharply increased the ratio of extracellular EGT concentration from 18.7% to 44.9%. Subsequently, an additional copy of egtABCDE, hisG, and mfsT1 was inserted into the genome with a site-specific genomic integration tool of M. neoaurum, leading a 2.7 times increase in EGT production. Co-enhancing the S-adenosyl-L-methionine regeneration pathway, or alternatively, the integration of three copies of egtABCDE, hisG and mfsT1 into the genome further increased the total EGT yield by 16.1% (64.6 mg/L) and 21.7% (67.7 mg/L), respectively. After 168-h cultivation, the highest titer reached 85.9 mg/L in the latter strain with three inserted copies. This study provided a solid foundation for genome engineering to increase the production of EGT in M. neoaurum.

Penile cancer (PC) is a rare male malignant tumor, with early lymph node metastasis and poor prognosis. Human papillomavirus (HPV) plays a key role in the carcinogenesis of PC. This review aims to summarize the association between HPV infection and PC in terms of virus-host genome integration patterns (the disrupted regions in the HPV and PC genome), genetic alterations, and epigenetic regulation (methylation and microRNA modification) occurring in HPV and PC DNA, as well as tumor immune microenvironment reprogramming. In addition, the potential of HPV vaccination strategies for PC prevention and treatment is discussed. Understanding of the HPV-related multidimensional mechanisms and the application of HPV vaccines will promote rational and novel management of PC.

Non-viral DNA donor template has been widely used for targeted genomic integration by homologous recombination (HR). This process has become more efficient with RNA guided endonuclease editor system such as CRISPR/Cas9. Circular single stranded DNA (cssDNA) has been harnessed previously as a genome engineering catalyst (GATALYST) for efficient and safe targeted gene knock-in. Here we developed enGager, a system with enhanced GATALYST associated genome editor, comprising a set of novel genome editors in which the integration efficiency of a circular single-stranded (css) donor DNA is elevated by directly tethering of the cssDNA to a nuclear-localized Cas9 fused with ssDNA binding peptides. Improvements in site-directed genomic integration and expression of a knocked-in DNA encoding GFP were observed at multiple genomic loci in multiple cell lines. The enhancement of integration efficiency, compared to unfused Cas9 editors, ranges from 1.5- to more than 6-fold, with the enhancement most pronounced for transgenes of > 4Kb in length in primary cells. enGager-enhanced genome integration prefers ssDNA donors which, unlike traditional dsDNA donors, are not concatemerized or rearranged prior to and during integration Using an enGager fused to an optimized cssDNA binding peptide, exceptionally efficient, targeted integration of the chimeric antigen receptor (CAR) transgene was achieved in 33% of primary human T cells. Enhanced anti-tumor function of these CAR-T primary cells demonstrated the functional competence of the transgenes. The \'tripartite editors with ssDNA optimized genome engineering\' (TESOGENASE™) systems help address the efficacy needs for therapeutic gene modification while avoiding the safety and payload size limitations of viral vectors currently used for CAR-T engineering.

Equine sarcoids (EqS) are fibroblast-derived skin tumors associated with bovine papillomavirus 1 and 2 (BPV-1 and -2). Based on Southern blotting, the BPV-1 genome was not found to be integrated in the host cell genome, suggesting that EqS pathogenesis does not result from insertional mutagenesis. Hence, CRISPR/Cas9 implies an interesting tool for selectively targeting BPV-1 episomes or genetically anchored suspected host factors. To address this in a proof-of-concept study, we confirmed the exclusive episomal persistence of BPV-1 in EqS using targeted locus amplification (TLA). To investigate the CRISPR/Cas9-mediated editing of BPV-1 episomes, primary equine fibroblast cultures were established and characterized. In the EqS fibroblast cultures, CRISPR-mediated targeting of the episomal E5 and E6 oncogenes as well as the BPV-1 long control region was successful and resulted in a pronounced reduction of the BPV-1 load. Moreover, the deletion of the equine Vimentin (VIM), which is highly expressed in EqS, considerably decreased the number of BPV-1 episomes. Our results suggest CRISPR/Cas9-based gene targeting may serve as a tool to help further unravel the biology of EqS pathogenesis.

Analyses of Illumina-based high-throughput sequencing data generated during characterization of the cotton leafroll dwarf virus population in Mississippi (2020-2022) consistently yielded contigs varying in size (most frequently from 4 to 7 kb) with identical nucleotide content and sharing similarities with reverse transcriptases (RTases) encoded by extant plant pararetroviruses (family Caulimoviridiae). Initial data prompted an in-depth study involving molecular and bioinformatic approaches to characterize the nature and origins of these caulimovirid-like sequences. As a result, here, we report on endogenous viral elements (EVEs) related to extant members of the family Caulimoviridae, integrated into a genome of upland cotton (Gossypium hirsutum), for which we propose the provisional name \"endogenous cotton pararetroviral elements\" (eCPRVE). Our investigations pinpointed a ~15 kbp-long locus on the A04 chromosome consisting of head-to-head orientated tandem copies located on positive- and negative-sense DNA strands (eCPRVE+ and eCPRVE-). Sequences of the eCPRVE+ comprised nearly complete and slightly decayed genome information, including ORFs coding for the viral movement protein (MP), coat protein (CP), RTase, and transactivator/viroplasm protein (TA). Phylogenetic analyses of major viral proteins suggest that the eCPRVE+ may have been initially derived from a genome of a cognate virus belonging to a putative new genus within the family. Unexpectedly, an identical 15 kb-long locus composed of two eCPRVE copies was also detected in a newly recognized species G. ekmanianum, shedding some light on the relatively recent evolution within the cotton family.

The isopentenol utilization pathway (IUP) is potential in terpenoids synthesis. This study aimed to construct IUP-employed Escherichia coli chassis for stably synthesizing terpenoids. As to effectiveness, promotor engineering strategy was employed to regulate IUP expression level, while ribosome-binding site (RBS) library of the key enzyme was constructed for screening the optimal RBS, followed by optimization of concentration of inducer and substrates, the titer of reporting production, lycopene, from 0.087 to 8.67 mg OD600 -1 . As about stability, the IUP expression cassette was integrated into the genome through transposition tool based on CRISPR-associated transposases. Results showed that the strain with 13 copies produced 1.78-fold lycopene titer that of the controlled strain with IUP-harbored plasmid, and it exhibited stable expression after ten successions while the plasmid loss was observed in the controlled strain in the 3rd succession. This strategy provides valuable information for rapid construction of highly effective and stable chassis employing IUP for terpenoids production.

Pterygium is one of the most frequent pathologies in ophthalmology, and is a benign, overgrowth of fibrovascular tissue, often with a wing-like appearance, from the conjunctiva over the cornea. It is composed of an epithelium and highly vascular, sub-epithelial, loose connective tissue. There is much debate surround the pathogenesis of pterygium and a number of theories have been put forward including genetic instability, cellular proliferation, inflammatory influence, and degeneration of connective tissue, angiogenesis, aberrant apoptosis and viral infection. At present, the involvement of human papillomavirus (HPV) in the genesis of pterygium is controversial, as have reported that HPV is present in 58% of cases, while others have failed to detect HPV in pterygium. In this study, we evaluated the presence and viral genotype of HPV DNA in pterygia and healthy conjunctiva sample, and virus integration into the cellular genome. Forty primary pterygia samples and 12 healthy conjunctiva samples were analyzed to HPV DNA presence by polymerase chain reaction, using MY09/MY11 primers of HPV-L1 gene. Viral genotype was identified by DNA sequence analysis of this amplicon. HPV integration into the cellular genome was analyzed by western blot detecting HPV-L1 capsid protein. Presence of HPV was observed in 19 of the 40 pterygia samples. In contrast, healthy conjunctiva samples were negative. To determine virus type, sequence analyses were performed. Interestingly, 11 out of the 19-pterygium samples were identified as HPV-11 type, meanwhile, the remaining 8 pterygium samples were identified as HPV-18. HPV-L1 capsid protein were found only in 3 out of the 10 samples studied. In conclusion, our study identified the presence of HPV DNA exclusively in pterygium samples and described HPV-11 and -18 genotypes. Our results suggest that HPV may be involved in the pathogenesis of pterygium. On the other hand, the expression of the L1-HPV protein suggests viral integration into the cellular genome.