Accelerated Screening of Alternative DNA Base-Organic Molecule-Base Architectures via Integrated Theory and Experiment.

Organic molecule-mediated noncanonical DNA self-assembly expands the standard DNA base-pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base-pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub-stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule-incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA-molecule architectures beyond Watson-Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA-based delivery system and probe design for sensitive detection of certain molecules.

The Mechanisms of miRNAs on Target Regulation and their Recent Advances in Atherosclerosis.

miRNAs are crucial regulators in a variety of physiological and pathological processes, while their regulation mechanisms were usually described as negatively regulating gene expression by targeting the 3'-untranlated region(3'-UTR) of target gene miRNAs through seed sequence in tremendous studies. However, recent evidence indicated the existence of non-canonical mechanisms mediated by binding other molecules besides mRNAs. Additionally, accumulating evidence showed that functions of intracellular and intercellular miRNAs exhibited spatiotemporal patterns. Considering that detailed knowledge of the miRNA regulating mechanism is essential for understanding the roles and further clinical applications associated with their dysfunction and dysregulation, which is complicated and not fully clarified. Based on that, we summarized the recently reported regulation mechanisms of miRNAs, including recognitions, patterns of actions, and chemical modifications. And we also highlight the novel findings of miRNAs in atherosclerosis progression researches to provide new insights for non-coding RNA-based therapy in intractable diseases.

Rational Design of Abasic Site-Containing DNA Triplexes To Bind Small Molecules with Low Nanomolar Dissociation Constants.

De novo design of functional biomacromolecules is of great interest to a wide range of fundamental science and technological applications, including understanding life evolution and biomacromolecular structures, developing novel catalysts, inventing medicines, and exploring high-performance materials. However, it is an extremely challenging task and its success is very limited. It requires a deep understanding of the relationships among the primary sequences, the 3D structures, and the functions of biomacromolecules. Herein, we report a rational, de novo design of a DNA aptamer that can bind melamine with high specificity and high affinity (dissociation constant Kd = 4.4 nM). The aptamer is essentially a DNA triplex, but contains an abasic site, to which the melamine binds. The aptamer-ligand recognition involves hydrogen-bonding, π-π stacking, and electrostatic interactions. This strategy has been further tested by designing aptamers to bind to guanosine. It is conceivable that such a rational strategy, with further development, would provide a general framework for designing functional DNA molecules.

Divergence and Convergence: Complexity Emerges in Crystal Engineering from an 8-mer DNA.

Biology provides plenty of examples on achieving complicated structures out of minimal numbers of building blocks. In contrast, structural complexity of designed molecular systems is achieved by increasing the numbers of component molecules. In this study, the component DNA strand assembles into a highly complex crystal structure via an unusual path of divergence and convergence. This assembly path suggests a route to minimalists for increasing structural complexity. The original purpose of this study is to engineer DNA crystals with high resolution, which is the primary motivation and a key objective for structural DNA nanotechnology. Despite great efforts in the last 40 years, engineered DNA crystals have not yet consistently reached resolution better than 2.5 Å, limiting their potential uses. Our research has shown that small, symmetrical building blocks generally lead to high resolution crystals. Herein, by following this principle, we report an engineered DNA crystal with unprecedented high resolution (2.17 Å) assembled from one single DNA component: an 8-base-long DNA strand. This system has three unique characteristics: (1) It has a very complex architecture, (2) the same DNA strand forms two different structural motifs, both of which are incorporated into the final crystal, and (3) the component DNA molecule is only an 8-base-long DNA strand, which is, arguably, the smallest DNA motif for DNA nanostructures to date. This high resolution opens the possibility of using these DNA crystals to precisely organize guest molecules at the Å level, which could stimulate a range of new investigations.

Engineering DNA Crystals toward Studying DNA-Guest Molecule Interactions.

Sequence-selective recognition of DNA duplexes is important for a wide range of applications including regulating gene expression, drug development, and genome editing. Many small molecules can bind DNA duplexes with sequence selectivity. It remains as a challenge how to reliably and conveniently obtain the detailed structural information on DNA-molecule interactions because such information is critically needed for understanding the underlying rules of DNA-molecule interactions. If those rules were understood, we could design molecules to recognize DNA duplexes with a sequence preference and intervene in related biological processes, such as disease treatment. Here, we have demonstrated that DNA crystal engineering is a potential solution. A molecule-binding DNA sequence is engineered to self-assemble into highly ordered DNA crystals. An X-ray crystallographic study of molecule-DNA cocrystals reveals the structural details on how the molecule interacts with the DNA duplex. In this approach, the DNA will serve two functions: (1) being part of the molecule to be studied and (2) forming the crystal lattice. It is conceivable that this method will be a general method for studying drug/peptide-DNA interactions. The resulting DNA crystals may also find use as separation matrices, as hosts for catalysts, and as media for material storage.

Case report: Complete response to pembrolizumab in a liver metastatic colon adenocarcinoma patient with a novel likely pathogenic germline MSH2 mutation.

Lynch syndrome (LS) is a genetic disorder mainly caused by germline mutations in mismatched repair (MMR) genes (MSH2, MLH1, MSH6, and PMS2) or deletions of the epithelial cell adhesion molecule gene (EPCAM). A 43-year-old Chinese male patient underwent radical surgery and was pathologically confirmed to have stage IIIB colon adenocarcinoma. After four cycles of standard adjuvant chemotherapy, the tumor reoccurred in situ with intestinal obstruction. The patient received secondary colectomy. Immunohistochemistry analysis revealed a loss of MSH2 protein expression in the surgical specimen. Noticing that the patient's mother and grandfather all were diagnosed with LS-related cancers, we collected the patient's and his mother's peripheral blood for genetic testing, and the result showed a six-base deletion of MSH2. Thus, we concluded that our patient had LS. Subsequently, the patient accepted pembrolizumab as the first-line systemic therapy after liver metastases. He achieved clinical complete response (cCR) within 2 months and remained progression-free for more than 2 years. The case report showed that MSH2 mutation (c.489_494deTGGGTA) is a likely pathogenic mutation, and immunotherapy (pembrolizumab) is effective for this patient.

LncRNA HCG18 promotes M2 macrophage polarization to accelerate cetuximab resistance in colorectal cancer through regulating miR-365a-3p/FOXO1/CSF-1 axis.

BACKGROUND: Cetuximab (CET) resistance in colorectal cancer (CRC) is responsible to poor prognosis to some extent. M2 macrophage polarization is closely correlated with drug resistance to cancers. Therefore, this study aims to investigate whether the mechanism of HCG18 on CET resistance to CRC involving in M2 macrophage polarization. METHODS: Clinic samples and SW620 cells with/without M0 macrophage co-culture served as experimental subjects. CET treatment was performed to induce SW620 cell resistant to CET. qRT-PCR and western blot were employed to evaluate the mRNA and protein expression of genes. The capabilities of cell viability, proliferation, migration and invasion were examined using CCK-8, clone formation assay and transwell. ELISA was employed to examine the protein concentrations of IL-10 and TGF-ß1. StarBase and luciferase activity assay were conducted to consolidate the interactions among HCG18, miR-365a-3p and FOXO1. RESULTS: In clinical samples and CRC cells, the abundance of HCG18 was enhanced whereas miR-365a-3p was reduced. Besides, HCG18 expression in CET-resistant tumor tissues was higher than that in CET-sensitive tumor tissues and the trend of miR-365a-3p was opposite to that of HCG18. HCG18 knockdown attenuated macrophage-induced CET resistance in SW620 cells and suppressed M2 polarization of THP-1 cells. Mechanistically, HCG18 interacted with miR-365a-3p and miR-365a-3p targeted FOXO1. MiR-365a-3p inhibitor abolished HCG18 knockdown-mediated inhibition of CET resistance, while FOXO1 knockdown compromised the influences of miR-365a-3p inhibitor. FOXO1 could positively regulate CSF-1 expression to promote M2 macrophage polarization and macrophage-induced CET resistance. CONCLUSION: Our results revealed that HCG18 promoted M2 macrophage polarization to facilitate CET resistance to CRC cells through modulating miR-365a-3p/FOXO1/CSF-1 axis.

Case Report: An Adolescent Soft Tissue Sarcoma With YWHAE-NUTM2B Fusion Is Effectively Treated With Combined Therapy of Epirubicin and Anlotinib.

Soft tissue sarcoma is a relatively rare entity that comprises heterogeneous types of tumors. Here we report the case of a 14-year-old girl with pelvic sarcoma with a YWHAE-NUTM2B fusion gene. This fusion transcript has been reported in endometrial stromal sarcomas and clear cell renal sarcomas, but its description in pelvic sarcomas is recent. To our knowledge, this is the first case report describing this translocation in an adolescent patient with soft tissue sarcoma. The patient underwent cytoreductive surgery, followed by systemic chemotherapy and targeted drug treatment. Surprisingly, the treatment was effective, and the young patient is being followed up in our department.

Regorafenib in Refractory Metastatic Colorectal Cancer: A Multi-Center Retrospective Study.

Background: Regorafenib improves progression-free survival (PFS) and overall survival (OS) in patients with refractory metastatic colorectal cancer (mCRC). Here, we report the treatment patterns of regorafenib in the third- or late-line setting for mCRC in four centers in China. Patients and Methods: Patients with refractory mCRC in four centers in China administered regorafenib from February 1, 2018 to June 31, 2021 were enrolled. Patients were grouped into 3 cohorts, namely, the monotherapy (regorafenib alone), chemo (regorafenib plus chemotherapy), and immune [regorafenib plus anti-PD1 (programmed cell death 1) antibodies] groups. Demographic, clinical, survival and safety data were retrospectively analyzed. Results: A total of 177 patients were included in this study. Of them, 116 (65.5%) were treated with regorafenib alone, while 28 (15.9%) and 33 (18.6%) were administered regorafenib plus chemotherapy and anti-PD1 antibodies, respectively. The median followed-up time was 9.2 months. The disease control rate (DCR) was 40.7%. The median PFS (mPFS) was 2.43 months and the median OS (mOS) was 12.2 months. The immune group had longer median PFS (3.5 m vs. 2.2 m, p = 0.043) compared with the monotherapy group. Patients administered regorafenib plus chemotherapy had longer median OS (15.9 m vs. 8.4 m, p = 0.032) compared with the monotherapy group. Patients who began regorafenib treatment at 120 mg had longer median PFS and OS compared with those who began at 80 mg (PFS: 3.7 m vs. 2.0 m; p <0.001; OS: 13.4 m vs. 10.2 m; p = 0.005). Patients with a final dose of 120 mg had longer median PFS and OS compared with the 80 mg or less group (PFS: 5.0 m vs. 2.3 m; p = 0.045; OS: UR (unreach) vs. 10.9 m; p = 0.003). There were 87.0% (154/177) patients who experienced AEs. Three groups had similar rates of AEs (86.2% vs. 89.3% vs. 87.9%; p = 0.89). Conclusion: Patients administered regorafenib alone or regorafenib in combination with other agents were relieved to some extent, with a disease control rate of 40.7%. Regorafenib plus anti-PD1 antibodies showed better PFS, while regorafenib plus chemotherapy had the most benefit in OS. There was no significant difference among three groups in terms of AEs.

Hepatic Artery Infusion Pump Combined With Systemic Chemotherapy for Patients With Liver Metastases From Breast Carcinoma.

Background: When liver metastasis in patients with breast cancer is diagnosed, treatment is generally palliative and usually consists of systemic therapies only. This study aimed to evaluate the efficacy and safety of hepatic arterial infusion (HAI) combined with systemic chemotherapy in patients with breast carcinoma liver metastases (BCLM). Methods: From January 2012 to December 2019, HAI catheter systems were implanted under the guide of digital subtract angiography (DSA) in 19 patients with BCLM. All patients received systemic chemotherapy and HAI gemcitabine plus floxuridine (FUDR). Methods: The overall response rate (ORR) of intrahepatic lesions was 73.7%, including 2 patients (10.5%) with complete remission (CR) and 12 patients (63.2%) with partial remission (PR). Additionally, we found that young patients (age < 55 years) had a higher ORR than the older (100% vs 44.4%, P = .011). The median overall survival (mOS) was 13.1 months. Kaplan-Meier survival curves demonstrated that the mOS was not significantly different between patients with < 9 intrahepatic lesions and those with ≥ 9 lesions (13.7 months vs 10.9 months, P = .225). The mOS was 14.3 and 10.6 months for patients without extrahepatic metastases and with extrahepatic metastases, respectively (P = .016). None of the patients had grade 4 toxicity. The grade 3 toxicities included leucopenia, neutropenia and diarrhea. Conclusions: HAI gemcitabine plus FUDR combined with systemic chemotherapy is effective in achieving a high local response and prolonging mOS for patients with BCLM and is associated with a relatively low rate of toxicity.

LncRNA HCG18 suppresses CD8+ T cells to confer resistance to cetuximab in colorectal cancer via miR-20b-5p/PD-L1 axis.

Aim: We aimed to explore the effect of long noncoding RNA HCG18 in colorectal cancer (CRC). Materials & methods: Relative gene and protein expression were screened. Colony formation and flow cytometry assays were performed to determine proliferation and apoptosis. Dual luciferase and RNA immunoprecipitation assays were conducted to validate the interaction between indicated molecules. Xenograft in nude mice was applied to verify the conclusion in vivo. Results:HCG18 and PD-L1 were upregulated while miR-20b-5p was downregulated in CRC tissue. Functional analysis revealed that lncRNA HCG18 promoted proliferation, migration and resistance to cetuximab of CRC cells via the miR-20b-5p/PD-L1 axis. Conclusion:HCG18 facilitated progress of the tumor, conferred to cetuximab resistance and suppressed CD8+ T cells via the miR-20b-5p/PD-L1 axis.

Lay abstract In the present study, we found a long noncoding RNA (lncRNA), HCG18 (a recently discovered lncRNA that facilitates tumor progression via multiple mechanisms), was upregulated in colorectal cancer (CRC). Further studies revealed that HCG18 suppressed CD8⁺ T-cell (cytotoxic T lymphocyte which kills cancer cell) activation to induce cetuximab (a first-line drug in CRC) resistance. Mechanically, HCG18 elevated expression of PD-L1 (a receptor in T-cell membranes, thus suppressing the proliferation of CD8⁺ cytotoxic T lymphocytes) via sponging (lncRNA binds with miRNA) miR-20b-5p. This study might provide a deeper insight into understanding cetuximab resistance in CRC.

Hsa_circ_0136666 activates Treg-mediated immune escape of colorectal cancer via miR-497/PD-L1 pathway.

PURPOSE: In the rankings of cancer mortality and incidence worldwide, colorectal cancer ranks fourth and the third, respectively. Circular RNA hsa_circ_0136666 (hsa_circ_0136666) is reported to participate in the growth of colorectal cancer. However, the mechanism by which hsa_circ_0136666 regulates the tumorigenesis of colorectal cancer needs to be further explored. In this study, we report here the role of hsa_circ_0136666 in the aberrant activation of Treg cells and immune evasion of tumor cells, providing a new strategy for the treatment of colorectal cancer. METHODS: Western blotting assay and qRT-PCR assay were used to determine protein and mRNA expression levels. Dual-luciferase reporter assay was used to evaluate the targeted regulatory relationship. RNA immunoprecipitation was used to detect RNA binding. Colony formation assay was utilized to measure the cell proliferation. Flow cytometry was used to assess cell apoptosis. Xenograft model was setup to evaluate tumor growth. RESULTS: The results showed that hsa_circ_0136666 and PD-L1 was increased in colorectal cancer cells while miR-497 was decreased in colorectal cancer cells when compared with normal colon epithelial cell line. Hsa_circ_0136666 was demonstrated to directly target miR-497, which also regulated PD-L1 by binding to its 3'UTR. Further mechanistic studies identified that hsa_circ_0136666 controlled cell proliferation and apoptosis via targeting miR-497 and regulating PD-L1 expression. Of note, hsa_circ_0136666 stimulated Treg cells mediated by miR-497/PD-L1 axis and its downstream signal pathway in Treg cells. Finally, hsa_circ_0136666 was found to accelerate the tumor growth in vivo. CONCLUSIONS: Our findings demonstrated that hsa_circ_0136666 promoted the expression of PD-L1 by inhibiting miR-497 level in colorectal cancer, thus inducing the activation of Treg cells and leading to the immune escape of tumor, providing a novel mechanistic insight into the pathogenesis of colorectal cancer.

Polθ reverse transcribes RNA and promotes RNA-templated DNA repair.

Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Šcrystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2'-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair.

Hepatic Artery Infusion of Floxuridine in Combination With Systemic Chemotherapy for Pancreatic Cancer Liver Metastasis: A Propensity Score-Matched Analysis in Two Centers.

AIM: To evaluate the efficacy of hepatic artery infusion (HAI) of floxuridine (FUDR) in combination with systemic chemotherapy in patients with pancreatic cancer liver metastases (PCLM). PATIENTS AND METHODS: We retrospectively collected clinical data of 347 patients with PCLM who underwent first-line chemotherapy at two Chinese centers between 2012 and 2019. Propensity score matching between patients with and without HAI was performed to compensate for differences in baseline characteristics. Objective response rate (ORR) and overall survival (OS) between groups were compared. HAI pump functionality was recorded. RESULTS: Data of 258 patients (62 patients with HAI and 196 patients without HAI) were used for matching. After 1:1 ratio matching, 62 patients per group were included. The intrahepatic ORR was 66.1% in the HAI group and 22.6% in the non-HAI group (P < 0.001), and the extrahepatic ORR was 25.0 versus 28.9% (P = 0.679). The median OS was significantly longer in HAI group (14.0 versus 10.8 months, P = 0.001). Multivariance COX regression showed HAI led to a decrease in hazard ratio for death by 61.8% (HR = 0.382; 95% CI: 0.252-0.578; P< 0.001). Subgroup analysis revealed that patients without EHM, with higher intrahepatic tumor burden and with synchronous liver metastasis benefited more from HAI. Dysfunction of HAI pump occurred in 5.7% of patients during the period of follow-up. CONCLUSIONS: In patients with PCLM, first-line treatment with HAI FUDR plus SCT resulted in higher intrahepatic response and better OS.

The hepatic microenvironment promotes lung adenocarcinoma cell proliferation, metastasis, and epithelial-mesenchymal transition via METTL3-mediated N6-methyladenosine modification of YAP1.

The inflammatory microenvironment plays an important role in the onset and progression of lung adenocarcinoma (LUAD), and the liver is a suitable site of metastasis for LUAD cells. However, whether the inflammatory microenvironment of the liver is conducive to the proliferation, invasion, and metastasis of LUAD cells remains unclear. In this study, we confirmed that the hepatic inflammatory microenvironment stimulated by IL-6 promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition of LUAD cells, increased the m6A methylation of total RNA, and transcriptionally activated METTL3 expression. Additionally, METTL3 activated the YAP1/TEAD signaling pathway by increasing the m6A modification and expression of YAP1 mRNA. These results indicate that the hepatic inflammatory microenvironment plays a role in regulating the biological functions of LUAD cells. Further, our study identifies a molecular mechanism that may provide a new strategy for the early diagnosis, treatment, and prognosis of liver metastasis in LUAD patients.

A poly(thymine)-melamine duplex for the assembly of DNA nanomaterials.

The diversity of DNA duplex structures is limited by a binary pair of hydrogen-bonded motifs. Here we show that poly(thymine) self-associates into antiparallel, right-handed duplexes in the presence of melamine, a small molecule that presents a triplicate set of the hydrogen-bonding face of adenine. X-ray crystallography shows that in the complex two poly(thymine) strands wrap around a helical column of melamine, which hydrogen bonds to thymine residues on two of its three faces. The mechanical strength of the thymine-melamine-thymine triplet surpasses that of adenine-thymine base pairs, which enables a sensitive detection of melamine at 3 pM. The poly(thymine)-melamine duplex is orthogonal to native DNA base pairing and can undergo strand displacement without the need for overhangs. Its incorporation into two-dimensional grids and hybrid DNA-small-molecule polymers highlights the poly(thymine)-melamine duplex as an additional tool for DNA nanotechnology.

Emergence of KRAS p.G13D mutation and acquired resistance to cetuximab in colorectal cancer with vulvar metastasis: A case report.

RATIONALE: Vulvar metastasis of colorectal cancer (CRC) and acquired resistance to cetuximab is a very rare phenomenon. To our knowledge, few cases have been reported in the English literatures. PATIENT CONCERNS: A 55-year-old woman was diagnosed as adenocarcinoma of the rectum and the primary tumor was detected to be Kirsten-RAS (KRAS) wild type. DIAGNOSES: The patient was diagnosed with rectal adenocarcinoma by colonoscopy. Positron emission tomography/computed tomography (PET-CT) revealed multiple lymph node and bone metastases. INTERVENTIONS: The patient received a first-line course of palliative chemotherapy with FOLFOX combined with cetuximab. OUTCOMES: After an initial response, acquired resistance to cetuximab occurred and vulvar metastasis was established by a second biopsy. Further molecular analysis showed that the KRAS mutation was detected in plasma samples and tumor tissues. LESSONS: Vulvar metastasis from CRC is relatively rare and indicates a poor prognosis. Routine physical examinations of cutaneous and subcutaneous may facilitate early detection of metastases and timely intervention of medical technology. Moreover, combining serial tumor biopsy, liquid biopsy, and radiologic imaging could help to define mechanisms of drug resistance and to guide selection of therapeutic strategies.

Making Engineered 3D DNA Crystals Robust.

Engineered 3D DNA crystals are promising scaffolds for bottom-up construction of three-dimensional, macroscopic devices from the molecular level. Nevertheless, this has been hindered by the highly constrained conditions for DNA crystals to be stable. Here we report a method to prepare robust 3D DNA crystals by postassembly ligation to remove this constraint. Specifically, sticky ends at crystal contacts were enzymatically ligated, and the covalent bonds significantly enhanced crystal stability, e.g., being stable at 65 °C. This method also enabled the fabrication of DNA crystals with complex architectures including crystal shell, core-shell, and matryoshka dolls. Furthermore, we have demonstrated the applications of the robust DNA crystals in biocatalysis and protein entrapment. Our study removes one key obstacle for the applications of DNA crystals and offers many new opportunities in DNA nanotechnology.