Genomic Disruption Research Articles

Photo-Enhanced CRISPR/Cas9 System Enables Robust PD-L1 Gene Disruption in Cancer Cells and Cancer Stem-Like Cells for Efficient Cancer Immunotherapy.

Blocking immune checkpoint pathways with an antibody or small interfering RNA (siRNA) has become a promising method to reactivate antitumor responses for cancer treatment. However, both blockade strategies achieve only temporary inhibition of these immune checkpoints. Herein, a photoswitched CRISPR/Cas9 system for genomic disruption of the PD-L1 gene is developed to achieve permanent blockade of the PD-1/PD-L1 pathway; this system is constructed by using a photoactivated self-degradable polyethyleneimine derivative and the plasmid pX330/sgPD-L1 (expression of the Cas9 protein and single-guide RNA targeting PD-L1). Under light irradiation, this photoswitched CRISPR/Cas9 system efficiently genetically disrupts the PD-L1 gene in not only bulk cancer cells but also cancer stem-like cells. As a result, the photoswitched CRISPR/Cas9 system significantly increases the infiltration of CD8+ T cells into tumor tissue, leading to effective activation of a T cell-mediated antitumor response against cancer cells and cancer stem-like cells. This study provides an alternative strategy to block the PD-1/PD-L1 pathway for efficacious immune checkpoint therapy.

Chromosomal instability in the prediction of pituitary neuroendocrine tumors prognosis

The purpose of this study was to analyze the impact of copy number variations (CNV) on sporadic pituitary neuroendocrine tumors (PitNETs) prognosis, to identify specific prognosis markers according to the known clinico-pathological classification. CGH array analysis was performed on 195 fresh-frozen PitNETs (56 gonadotroph, 11 immunonegative, 56 somatotroph, 39 lactotroph and 33 corticotroph), with 5 years post-surgery follow-up (124 recurrences), classified according to the five-tiered grading classification (invasion, Ki-67, mitotic index and p53 positivity). Effect of alterations on recurrence was studied using logistic regression models. Transcriptomic analysis of 32 lactotroph tumors was performed. The quantity of CNV was dependent on tumor type: higher in lactotroph (median(min–max) = 38% (0–97) of probes) compared to corticotroph (11% (0–77)), somatotroph (5% (0–99)), gonadotroph (0% (0–10)) and immunonegative tumors (0% (0–17). It was not predictive of recurrence in the whole cohort. In lactotroph tumors, genome instability, especially quantity of gains, significantly predicted recurrence independently of invasion and proliferation (p-value = 0.02, OR = 1.2). However, no specific CNV was found as a prognostic marker. Transcriptomic analysis of the genes included in the CNV and associated with prognosis didn’t show significantly overrepresented pathway. In somatotroph and corticotroph tumors, USP8 and GNAS mutations were not associated with genome disruption or recurrence respectively. To conclude, CGH array analysis showed genome instability was dependent on PitNET type. Lactotroph tumors were highly altered and the quantity of altered genome was associated with poorer prognosis though the mechanism is unclear, whereas gonadotroph and immunonegative tumors showed the same ‘quiet’ profile, leaving the mechanism underlying tumorigenesis open to question.

Abstract CT052: Clinical safety and efficacy study of TruUCAR™ GC027: The first-in-human, universal CAR-T therapy for adult relapsed/refractory T-cell acute lymphoblastic leukemia (r/r T-ALL)

Abstract Introduction: Patients with T cell malignancies usually have high relapse and mortality rates. Due to shared common surface antigen and potential contamination by malignant cells, development of CAR-T therapies for r/r T-ALL has been lagged, regardless of the costly and lengthy process of autologous CAR-T production. To overcome these challenges, we developed a universal CAR-T platform (TruUCAR™) that displayed superior expansion in patients without using preconditioning biologics such as αCD52 antibody. Here we report results from a prospective study of GC027, the first-in-human, universal CAR-T therapy for treating adult patients with r/r T-ALL to evaluate the safety and clinical efficacy. Methods: TruUCART™ GC027 contains a second-generation CAR with genomic disruption of TCRα and CD7 by CRISPR/Cas9 system to avoid GvHD and fratricide. It is manufactured using lentivirus and leukopaks from HLA-mismatched healthy donors. A T-ALL xenograft murine model were used to assess anti-leukemic efficacy and expansion. Preliminary safety, anti-leukemic activity and expansion kinetics of GC027 are being evaluated in in a single-arm, open-label, multi-center, prospective study for treating adult r/r T-ALL. To date, a total of 5 patients (age 19-38 yrs, median 24 yrs) were enrolled with marrow tumor load 4-80.2% (median prior lines 5). All 5 pts have received a 6-day enhanced preconditioning chemotherapy followed by a single infusion of GC027. Adverse events, disease response, and expansion kinetics were evaluated in this study. Results: GC027 demonstrated robust anti-leukemic activity and expansion in a highly malignant CCRF-CEM xenograft murine model. All mice infused with GC027 exhibited significantly reduced tumor burden and prolonged survival compared to control groups. As of Feb. 6, 2020, 5 pts had received a single dose of GC027, including 1 at 0.6x107/kg, 3 at 1x107/kg and 1 at 1.5x107/kg. 4 pts achieved MRD negative complete responses (MRD- CR) at D28 evaluation: 3 of them remained MRD- at follow-up re-evaluations (D118, 61, 161, respectively, and none was bridged to HSCT); 1 just achieved MRD- CR at D28 and follow-up results will be updated at the meeting. 1 pt achieved MRD+ CR at D14 but had relapsed disease at D29. In all 4 pts with MRD- CR, peak expansions of GC027 in peripheral blood were observed between week 1-2. In 1 pt with CNS disease, GC027 was detected in specimens from his bone marrow and cerebrospinal fluid (CSF). 4 pts experienced Grade 3 cytokine release syndrome (CRS) and 1 pt had Grade 4 CRS (by ASBMT Consensus Grading) along with elevated levels of IL6, IFNγ and TNFα. CRS symptoms were manageable and resolved after treatment and supportive care. None developed neurotoxicity or GvHD. 1 pt had prolonged cytopenia due to fungal infection and required anti-fungal therapy. Conclusions: With a single infusion of GC027, 80% of the patients had robust CAR-T cell expansion and achieved persistent MRD- CR without using any biologics as part of the preconditioning therapy or bridging to HSCT. As the first-in-human, universal CAR-T therapy for adult r/r T-ALL, GC027 has demonstrated superior clinical efficacy and induced deep response in patients with acceptable safety profile. The trial enrollment is ongoing and updated data will be presented at the meeting. Citation Format: Xinxin Wang, Shiqi Li, Lei Gao, Zhongtao Yuan, Kun Wu, Lin Liu, Le Luo, Yao Liu, Cheng Zhang, Jia Liu, Chunhui Yang, Yu Li, Zhimin Li, Jiaping He, Duanpeng Wang, Xun Ye, Xu Tan, Ruihao Huang, Jianning Ge, Yu Han, Dingsong Zhang, Youcheng Wang, Lihua Fang, Yingnian Chen, Wei Cao, Sanbin Wang, Xi Zhang. Clinical safety and efficacy study of TruUCAR™ GC027: The first-in-human, universal CAR-T therapy for adult relapsed/refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT052.

Field cricket genome reveals the footprint of recent, abrupt adaptation in the wild.

Evolutionary adaptation is generally thought to occur through incremental mutational steps, but large mutational leaps can occur during its early stages. These are challenging to study in nature due to the difficulty of observing new genetic variants as they arise and spread, but characterizing their genomic dynamics is important for understanding factors favoring rapid adaptation. Here, we report genomic consequences of recent, adaptive song loss in a Hawaiian population of field crickets (Teleogryllus oceanicus). A discrete genetic variant, flatwing, appeared and spread approximately 15 years ago. Flatwing erases sound‐producing veins on male wings. These silent flatwing males are protected from a lethal, eavesdropping parasitoid fly. We sequenced, assembled and annotated the cricket genome, produced a linkage map, and identified a flatwing quantitative trait locus covering a large region of the X chromosome. Gene expression profiling showed that flatwing is associated with extensive genome‐wide effects on embryonic gene expression. We found that flatwing male crickets express feminized chemical pheromones. This male feminizing effect, on a different sexual signaling modality, is genetically associated with the flatwing genotype. Our findings suggest that the early stages of evolutionary adaptation to extreme pressures can be accompanied by greater genomic and phenotypic disruption than previously appreciated, and highlight how abrupt adaptation might involve suites of traits that arise through pleiotropy or genomic hitchhiking.

Factors Impacting Efficacy of AAV-Mediated CRISPR-Based Genome Editing for Treatment of Choroidal Neovascularization

Frequent injections of anti-vascular endothelial growth factor (anti-VEGF) agents are a clinical burden for patients with neovascular age-related macular degeneration (AMD). Genomic disruption of VEGF-A using adeno-associated viral (AAV) delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 has the potential to permanently suppress aberrant angiogenesis, but the factors that determine the optimal efficacy are unknown. Here, we investigate two widely used Cas9 endonucleases, SpCas9 and SaCas9, and evaluate the relative contribution of AAV-delivery efficiency and genome-editing rates in vivo to determine the mechanisms that drive successful CRISPR-based suppression of VEGF-A, using a mouse model of laser-induced choroidal neovascularization (CNV). We found that SpCas9 demonstrated higher genome-editing rates, greater VEGF reduction, and more effective CNV suppression than SaCas9, despite similar AAV transduction efficiency between a dual-vector approach for SpCas9 and single-vector system for SaCas9 to deliver the Cas9 orthologs and single guide RNAs (gRNAs). Our results suggest that successful VEGF knockdown using AAV-mediated CRISPR systems may be determined more by the efficiency of genome editing rather than viral transduction and that SpCas9 may be more effective than SaCas9 as a potential therapeutic strategy for CRISPR-based treatment of CNV in neovascular AMD.

The papillomavirus E5 gene does not affect EGFR transcription and overall survival in cervical cancer.

The human papillomavirus (HPV) E5 gene encodes a small and highly hydrophobic oncoprotein that affects immune evasion, cell proliferation, loss of apoptotic capacity and angiogenesis in tumors. E5 shows an affinity for biological membranes and was associated with an increase of epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) signaling through the accumulation of EGFR in cellular membranes. Due to the frequent integration of the HPV genome into the host cell genome, E5 is frequently not transcribed in cervical tumors. In this study we looked forward to verifying whether the potential expression of E5 protein inhuman papillomavirus 16 positive (HPV16+ ) andhuman papillomavirus 18 positive (HPV18+ ) cervical tumors was associated with levels of EGFR and vascular endothelial growth factor A (VEGFA) transcription and with patients overall survival. Association between the presence of E5 transcripts and viral genome disruption was observed for HPV16+ and HPV18+ tumors. Association was not observed between tumors potentially capable of translating E5 and EGFR or VEGFA transcriptional levels. Similarly, the capability of translating E5 and overall survival in patients with HPV16+ squamous cell carcinoma tumors stage ≥ IB2 were not associated. The likely presence of E5 transcripts was neither associated to a higher activity of the EGFR-VEGFA pathway nor to the overall survival of patients with HPV16+ squamous cell carcinoma in stages ≥ IB2.

Helicases FANCJ, RTEL1 and BLM Act on Guanine Quadruplex DNA in Vivo.

Guanine quadruplex (G4) structures are among the most stable secondary DNA structures that can form in vitro, and evidence for their existence in vivo has been steadily accumulating. Originally described mainly for their deleterious effects on genome stability, more recent research has focused on (potential) functions of G4 structures in telomere maintenance, gene expression, and other cellular processes. The combined research on G4 structures has revealed that properly regulating G4 DNA structures in cells is important to prevent genome instability and disruption of normal cell function. In this short review we provide some background and historical context of our work resulting in the identification of FANCJ, RTEL1 and BLM as helicases that act on G4 structures in vivo. Taken together these studies highlight important roles of different G4 DNA structures and specific G4 helicases at selected genomic locations and telomeres in regulating gene expression and maintaining genome stability.

Antimicrobial Activity and Proposed Action Mechanism of 3-Carene against Brochothrix thermosphacta and Pseudomonas fluorescens.

3-Carene is an antimicrobial monoterpene that occurs naturally in a variety of plants and has an ambiguous antibacterial mechanism against food-borne germs. The antibacterial effects and action mechanism of 3-carene against Gram-positive Brochothrix thermosphacta ACCC 03870 and Gram-negative Pseudomonas fluorescens ATCC 13525 were studied. Scanning electron microscopy (SEM) examination and leakage of alkaline phosphatase (AKP) verified that 3-carene caused more obvious damage to the morphology and wall structure of B. thermosphacta than P. fluorescens. The release of potassium ions and proteins, the reduction in membrane potential (MP), and fluorescein diacetate (FDA) staining further confirmed that the loss of the barrier function of the cell membrane and the leakage of cytoplasmic contents were due to the 3-carene treatment. Furthermore, the disorder of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), pyruvate kinase (PK), and ATP content indicated that 3-carene could lead to metabolic dysfunction and inhibit energy synthesis. In addition, the results from the fluorescence analysis revealed that 3-carene could probably bind to bacterial DNA and affect the conformation and structure of genomic DNA. These results revealed that 3-carene had strong antibacterial activity against B. thermosphacta and P. fluorescens via membrane damage, bacterial metabolic perturbations, and genomic DNA structure disruption, interfering in cellular functions and even causing cell death.

Genetic Ablation of the Cystine Transporter xCT in PDAC Cells Inhibits mTORC1, Growth, Survival, and Tumor Formation via Nutrient and Oxidative Stresses.

Although chemoresistance remains a primary challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC), exploiting oxidative stress might offer novel therapeutic clues. Here we explored the potential of targeting cystine/glutamate exchanger (SLC7A11/xCT), which contributes to the maintenance of intracellular glutathione (GSH). Genomic disruption of xCT via CRISPR-Cas9 was achieved in two PDAC cell lines, MiaPaCa-2 and Capan-2, and xCT-KO clones were cultivated in the presence of N-acetylcysteine. Although several cystine/cysteine transporters have been identified, our findings demonstrate that, in vitro, xCT plays the major role in intracellular cysteine balance and GSH biosynthesis. As a consequence, both xCT-KO cell lines exhibited amino acid stress with activation of GCN2 and subsequent induction of ATF4, inhibition of mTORC1, proliferation arrest, and cell death. Tumor xenograft growth was delayed but not suppressed in xCT-KO cells, which indicated both the key role of xCT and also the presence of additional mechanisms for cysteine homeostasis in vivo. Moreover, rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling. These two hallmarks of ferroptotic cell death were prevented by vitamin E or iron chelation. Finally, in vitro pharmacologic inhibition of xCT by low concentrations of erastin phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in PDAC cell lines. In conclusion, our findings strongly support that inhibition of xCT, by its dual induction of nutritional and oxidative cellular stresses, has great potential as an anticancer strategy. SIGNIFICANCE: The cystine/glutamate exchanger xCT is essential for amino acid and redox homeostasis and its inhibition has potential for anticancer therapy by inducing ferroptosis.

Abstract 2868: CD99 inhibition by clofarabine induces Ewing sarcoma cytotoxicity through activation of FGFR1 and downstream kinase pathways

Abstract Ewing sarcoma (ES) cells express high levels of the cell surface protein CD99, which is used for diagnosis of ES. More importantly, the inhibition of CD99 activity results in reduced growth of ES cells in vitro and in vivo. In an earlier publication, we have identified clofarabine and cladribine as selective inhibitors of CD99, which inhibited ES growth both in vitro and in vivo. Clofarabine and cladribine directly bound to CD99, inhibited its molecular interactions, and regulated CD99 specific intracellular signaling pathways. In order to gain further insight into how clofarabine may regulate cellular functions through inhibiting CD99, we utilized a phospho-kinase array to identify changes in phosphorylation levels of 43 proteins in response to clofarabine or CD99 antibody treatment. We identified ERK1/2-MSK1/2-CREB signaling axis as one of the signaling pathways downstream of CD99. These findings were validated in four different ES cell lines and in xenograft lysates that were harvested from clofarabine-treated mice compared to control mice. The phosphorylation events induced by clofarabine treatment was significantly diminished when CD99 was knocked down either by siRNA-mediated knockdown or CRISPR/Cas9-mediated genomic disruption. Time-course experiments on ES cells showed that clofarabine triggers a very rapid signaling cascade through CD99. Cytarabine, a structurally similar pyrimidine analog and an inhibitor of the EWS-FLI1 transcriptional activity that has been failed in clinical trials on ES patients, did not activate ERK1/2, MSK1/2 or CREB phosphorylation in ES cells. In order to test whether the observed changes in phosphorylation levels are required for cell death, we screened a kinase inhibitor small molecule library for their ability to rescue clofarabine-induced death of ES cells. We identified TG100-115 (PI3K inhibitor) and rebastinib (c-abl inhibitor) as the most potent kinase inhibitors that showed significant reversal of clofarabine-induced ES cell death, suggesting that clofarabine-induced death of ES cells requires specific phosphorylation cascades. We then discovered that CD99 can be co-immunoprecipitated with FGFR1 and clofarabine treatment of ES cells results in phosphorylation of FGFR1. In conclusion, we discovered a novel mechanism of action for clofarabine and cladribine in that their selective cytotoxic effect on ES is through inhibiting CD99 on the cell surface, and it is not dependent on their ability to inhibit DNA synthesis. CD99 inhibition resulted in increased phosphorylation of FGFR and two downstream signaling pathways (PI3K/Akt and MEK1/ERK1). Inhibition of PI3K pathway rescues clofarabine-induced ES cell cytotoxicity. These findings provide additional mechanistic support for repurposing clofarabine and cladribine for ES indication. Note: This abstract was not presented at the meeting. Citation Format: Haydar Celik, Levent Dusunceli, Anna Molotkova, David V. Allegakoen, Erin J. Conn, Jeff R. Petro, Jeffrey A. Torestky, Aykut Uren. CD99 inhibition by clofarabine induces Ewing sarcoma cytotoxicity through activation of FGFR1 and downstream kinase pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2868.

A familial chromosomal complex rearrangement confirms RUNX1T1 as a causative gene for intellectual disability and suggests that 1p22.1p21.3 duplication is likely benign

BackgroundComplex chromosomal rearrangements are constitutive structural aberrations involving three or more breaks. They can be balanced or unbalanced and result in different outcomes, depending on deletion/duplication of genomic material, gene disruption, or position effects.Case presentationWe report on a patient presenting with severe anemia, splenomegaly, mild intellectual disability and facial dysmorphisms harboring a 4.3 Mb duplication at 1p22.1p21.3 and a 2.1 Mb deletion at 8q21.3q22.1, involving RUNX1T1 gene. The healthy brother presented the same duplication of chromosome 1p as at 1p22.1p21.3.ConclusionsThe rearrangement found both these siblings resulted from malsegregation in the proband and recombination in her healthy brother of a balanced paternal complex chromosomal rearrangement. These results confirm RUNX1T1 as a causative gene for intellectual disability and suggest the 1p22.1p21.3 duplication is likely benign.

Identification of the gene cluster for bistropolone-humulene meroterpenoid biosynthesis in Phoma sp.

Eupenifeldin, a bistropolone meroterpenoid, was first discovered as an antitumor agent from the fungus Eupenicillium brefeldianum. We also isolated this compound and a new congener from a strain of Phoma sp. (CGMCC 10481), and evaluated their antitumor effects. Eupenifeldin showed potent in vitro anti-glioma activity. This tropolone-humulene-tropolone meroterpenoid could be originated from two units of tropolone orthoquinone methides and a 10-hydroxyhumulene moiety via hetero-Diels-Alder reactions. To explore the biosynthesis of this class of tropolonic sesquiterpenes, the genome of a eupenifeldin-producing Phoma sp. was sequenced and analyzed. The biosynthetic gene cluster of eupenifeldin (eup) was identified and partially validated by genomic analysis, gene disruption, and product analysis. A nonreducing polyketide synthase EupA, a FAD-dependent monooxygenase EupB, and a non-heme Fe (II)-dependent dioxygenase EupC, were identified as the enzymes responsible for tropolone formation. While the terpene cyclase EupE of an unknown family was characterized to catalyze humulene formation, and a cytochrome P450 enzyme EupD was responsible for hydroxylation of humulene. This study sheds light on the biosynthesis of eupenifeldin, and paves the way to further decipher its biosynthetic pathway.

TRPC6 inactivation does not affect loss of renal function in nephrotoxic serum glomerulonephritis in rats, but reduces severity of glomerular lesions

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in a variety of chronic kidney diseases including familial and acquired forms of focal and segmental glomerulosclerosis (FSGS) and renal fibrosis following ureteral obstruction. Here we have examined the role of TRPC6 in progression of inflammation and fibrosis in the nephrotoxic serum (NTS) model of crescentic glomerulonephritis. This was assessed in rats with non-functional TRPC6 channels due to genomic disruption of an essential domain in TRPC6 channels (Trpc6del/del rats) and wild-type littermates (Trpc6wt/wt rats). Administration of NTS evoked albuminuria and proteinuria observed 4 and 28 days later that was equally severe in Trpc6wt/wt and Trpc6del/del rats. By 28 days, there were dense deposits of complement and IgG within glomeruli in both genotypes, accompanied by severe inflammation and fibrosis readily observed by standard histological methods, and also by increases in renal cortical expression of multiple markers (α-smooth muscle actin, vimentin, NLRP3, and CD68). Tubulointerstitial fibrosis appeared equally severe in Trpc6wt/wt and Trpc6del/del rats. TRPC6 inactivation did not protect against the substantial declines in renal function (increases in blood urea nitrogen, serum creatinine and kidney:body weight ratio) in NTS-treated animals, and increases in a urine maker of proximal tubule pathology (β2-macroglobulin) were actually more severe in Trpc6del/del animals. By contrast, glomerular pathology, blindly scored from histology, and from renal cortical expression of podocin suggested a partial but significant protective effect of TRPC6 inactivation within the glomerular compartment, at least during the autologous phase of the NTS model.

HACE1 is a potential tumor suppressor in osteosarcoma

Osteosarcoma is a malignant bone sarcoma characterized by extensive genomic disruption and a propensity for metastatic spread. Osteoid production suggests a close relationship with normal osteoblasts, and the latter are the presumptive cell of origin of this disease. The HACE1 gene, localized to human chromosome 6q21, encodes the HACE1 HECT E3 ligase, a tumor suppressor in diverse tumors that acts in part by targeting the activated form of RAC1 GTPase for proteasomal degradation. Disruption or loss of 6q21 is relatively common in osteosarcomas, and Hace1−/−/Tp53+/− mice frequently develop osteosarcomas, in contrast to Tp53+/− mice, which do not. This suggests an unexplored link between HACE1 loss and osteosarcoma. Here we compared HACE1 expression in normal osteoblasts and osteosarcoma cell lines in vitro by western blotting and quantitative RT-PCR, and in human osteosarcoma specimens by immunohistochemistry. Both HACE1 transcript and protein levels were reduced in osteosarcoma compared to osteoblasts in vitro. Reduced HACE1 expression in osteosarcoma tumors was observed in 76% of cases and associated with high-grade lesions. Further, clonally derived pairs of high and low metastatic osteosarcoma cell lines showed significant downregulation in the high compared to corresponding low metastatic cells. Ectopic expression of HACE1 markedly inhibited anchorage-independent growth and cell motility of HACE1 osteosarcoma cell lines, and was associated with reduced RAC1 activation and decreased reactive oxygen species (ROS). Finally, HACE1 overexpression blocked osteosarcoma xenograft growth and dramatically reduced pulmonary metastases. These findings point to a potential tumor suppressor function for HACE1 in osteosarcoma.

Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems.

Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors – HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae – robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.

Copy number variation profile in noninvasive prenatal testing (NIPT) can identify co-existing maternal malignancies: Case reports and a literature review

ObjectiveThe coexistence of maternal malignancy and pregnancy has received increasing attention in Noninvasive prenatal testing (NIPT) studies. Malignancy in pregnant women potentially affects the copy number variation (CNV) profile in NIPT results. Only one case of hematologic cancer has been reported in a Hong-Kong pregnant women, and solid tumors have never been reported in pregnant Chinese women. Case reportThe patients with dysgerminoma and cervical cancer showed aberrant chromosomal aneuploidies in NIPT and concordant patterns of genome disruption in tumor tissues. The genomic aberrations in the gastric cancer patient had similar copy number variation pattern of gastric cancer. ConclusionThe findings in this study and the literature review further validate the effect of maternal malignancy on the copy number variation profile in NIPT data and strengthen the possibility of detecting malignant tumors with NIPT in the future.

Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections.

The CRISPR/Cas9 system has been applied in the genome editing and disruption of latent infections for herpesviruses such as the herpes simplex virus, Epstein–Barr virus, cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus. CRISPR/Cas9-directed mutagenesis can introduce similar types of mutations to the viral genome as can bacterial artificial chromosome recombination engineering, which maintains and reconstitutes the viral genome successfully. The cleavage mediated by CRISPR/Cas9 enables the manipulation of disease-associated viral strains with unprecedented efficiency and precision. Additionally, current therapies for herpesvirus productive and latent infections are limited in efficacy and cannot eradicate viruses. CRISPR/Cas9 is potentially adapted for antiviral treatment by specifically targeting viral genomes during latent infections. This review, which focuses on recently published progress, suggests that the CRISPR/Cas9 system is not only a useful tool for basic virology research, but also a promising strategy for the control and prevention of herpesvirus latent infections.

HPV DNA methylation at the early promoter and E1/E2 integrity: A comparison between HPV16, HPV18 and HPV45 in cervical cancer

ObjectivesTo compare and describe type-specific characteristics of HPV16, HPV18 and HPV45 in cervical cancer with respect to 3′LCR methylation and disruption of E1/E2. MethodsThe methylation level of 137 cervical cancer samples (70 with HPV16, 37 with HPV18, and 30 with HPV45) of Brazilian patients was analyzed by pyrosequencing. PCR amplifications were performed to characterize E1 and E2 disruption as an episomal surrogate. ResultsThe 3′LCR of HPV16 showed a higher methylation at all CpG sites (7%, 9%, 11%, 10% and 10%) than homologous HPV18 regions (4%, 5%. 6%, 9% and 5%) and HPV45 regions (7%, 7% and 5%). Presence of intact E1/E2 was associated with higher HPV16 and HPV18 methylation levels at all CpG sites (p < 0.05). Disruption of E1/E2 was more frequently found in HPV45 (97%) and HPV18 (84%) than in HPV16 DNA (30%). HPV16 disruption was more frequently found in E1 (48%) unlike HPV18, where it was found in E2 (61%). Concomitant disruption of E1/E2 was most frequent in HPV45 (72%). ConclusionsThe findings showed a higher methylation associated with intact E1/E2 for HPV16 and HPV18. The closely phylogenetic related HPV18 and HPV45 share a similar methylation level and the frequency of viral genome disruption.

Applications of CRISPR/Cas System to Bacterial Metabolic Engineering.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli. Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.

Prevalence of anal infection due to high-risk human papillomavirus and analysis of E2 gene integrity among women with cervical abnormalities

BackgroundHigh-risk human papillomaviruses (HR-HPV) infection has been associated with 90% of anal cancer cases. Women with abnormal cytology are a high-risk group to develop anal neoplasia. The aim of this study is to describe the prevalence and epidemiology of HR-HPV 16, 18, 45, and 58 anal infections in women with cervical abnormalities, as well as to assess E2 gene integrity. MethodsA cross-sectional study was performed on 311 cervical and 311 anal samples from patients with abnormal cytology in two colposcopy clinics in Yucatan, Mexico. A specific PCR for oncogenes was performed in order to identify HVP 16, 18, 45 and 58. Real time PCR was used to amplify the whole HPV 16, 18, and 58 E2 gene to verify its integrity in anal samples. ResultsHigh risk HPV 16, 18, 58, and/or 45 were found in 41.47% (129/311) of cervical samples, and in 30.8% (96/331) of anal samples, with 18% (57/311) of the patients being positive in both samples. The same genotypes in both anatomical sites were observed in 11.25% (35/311). The E2 gene was disrupted in 82% of all tested samples. The frequency of genome disruption viral integration in anal samples by genotype was: HPV 58 (97.2%); HPV 16 (72.4%), and HPV 18 (0%). ConclusionWomen with cervical disease have HR-HPV anal infections, and most of them have the E2 gene disrupted, which represents a risk to develop anal cancer.