pH-Triggered Controlled Release of Chlorhexidine Using Chitosan-Coated Titanium Silica Composite for Dental Infection Prevention.

Peri-implantitis is a growing pathological concern for dental implants which aggravates the occurrence of revision surgeries. This increases the burden on both hospitals and the patients themselves. Research is now focused on the development of materials and accompanying implants designed to resist biofilm formation. To enhance this endeavor, a smart method of biofilm inhibition coupled with limiting toxicity to the host cells is crucial. Therefore, this research aims to establish a proof-of-concept for the pH-triggered release of chlorhexidine (CHX), an antiseptic commonly used in mouth rinses, from a titanium (Ti) substrate to inhibit biofilm formation on its surface. To this end, a macroporous Ti matrix is filled with mesoporous silica (together referred to as Ti/SiO2), which acts as a diffusion barrier for CHX from the CHX feed side to the release side. To limit release to acidic conditions, the release side of Ti/SiO2 is coated with crosslinked chitosan (CS), a pH-responsive and antimicrobial natural polymer. Scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM/EDX) and Fourier transform infrared (FTIR) spectroscopy confirmed successful CS film formation and crosslinking on the Ti/SiO2 disks. The presence of the CS coating reduced CHX release by 33% as compared to non-coated Ti/SiO2 disks, thus reducing the antiseptic exposure to the environment in normal conditions. Simultaneous differential scanning calorimetry and thermogravimetric analyzer (SDT) results highlighted the thermal stability of the crosslinked CS films. Quartz crystal microbalance with dissipation monitoring (QCM-D) indicated a clear pH response for crosslinked CS coatings in an acidic medium. This pH response also influenced CHX release through a Ti/SiO2/CS disk where the CHX release was higher than the average trend in the neutral medium. Finally, the antimicrobial study revealed a significant reduction in biofilm formation for the CS-coated samples compared to the control sample using viability quantitative polymerase chain reaction (v-qPCR) measurements, which were also corroborated using SEM imaging. Overall, this study investigates the smart triggered release of pharmaceutical agents aimed at inhibiting biofilm formation, with potential applicability to implant-like structures.

RAD54L2-mediated DNA damage avoidance pathway specifically preserves genome integrity in response to topoisomerase 2 poisons.

Type II topoisomerases (TOP2) form transient TOP2 cleavage complexes (TOP2ccs) during their catalytic cycle to relieve topological stress. TOP2ccs are covalently linked TOP2-DNA intermediates that are reversible but can be trapped by TOP2 poisons. Trapped TOP2ccs block transactions on DNA and generate genotoxic stress, which are the mechanisms of action of TOP2 poisons. How cells avoid TOP2cc accumulation remains largely unknown. In this study, we uncovered RAD54 like 2 (RAD54L2) as a key factor that mediates a TOP2-specific DNA damage avoidance pathway. RAD54L2 deficiency conferred unique sensitivity to treatment with TOP2 poisons. RAD54L2 interacted with TOP2A/TOP2B and ZATT/ZNF451 and promoted the turnover of TOP2 from DNA with or without TOP2 poisons. Additionally, inhibition of proteasome activity enhanced the chromatin binding of RAD54L2, which in turn led to the removal of TOP2 from chromatin. In conclusion, we propose that RAD54L2-mediated TOP2 turnover is critically important for the avoidance of potential TOP2-linked DNA damage under physiological conditions and in response to TOP2 poisons.

TDP1-independent pathways in the process and repair of TOP1-induced DNA damage.

Anticancer drugs, such as camptothecin (CPT), trap topoisomerase I (TOP1) on DNA and form TOP1 cleavage complexes (TOP1cc). Alternative repair pathways have been suggested in the repair of TOP1cc. However, how these pathways work with TDP1, a key repair enzyme that specifically hydrolyze the covalent bond between TOP1 catalytic tyrosine and the 3'-end of DNA and contribute to the repair of TOP1cc is poorly understood. Here, using unbiased whole-genome CRISPR screens and generation of co-deficient cells with TDP1 and other genes, we demonstrate that MUS81 is an important factor that mediates the generation of excess double-strand breaks (DSBs) in TDP1 KO cells. APEX1/2 are synthetic lethal with TDP1. However, deficiency of APEX1/2 does not reduce DSB formation in TDP1 KO cells. Together, our data suggest that TOP1cc can be either resolved directly by TDP1 or be converted into DSBs and repaired further by the Homologous Recombination (HR) pathway.

A link between DNA double-strand breaks and regulation of global translation.

In response to double-strand breaks (DSBs) in the DNA, cells undergo transcriptional, translational and post-translational reprogramming to tackle the damage. In this study by Riepe et al., the authors have shown that the global translation inhibition of proteins is concomitant to DNA damage response. Treatment with various DSB-generating agents can cause a major downregulation in the translation of cellular proteins except for the ISR (integrated stress response) proteins. Authors report a specific and significant reduction in the level of a core ribosomal RPS27A protein coupled to kinetics of DSB induction and repair. The study proposes that molecular alterations generated as a by-product of DNA damage may inadvertently impact phenotypic responses of the cells and a cautious approach must be followed when utilizing DSB-based genome editing techniques. Comment on: https://doi.org/10.1111/febs.16321.

MicroRNA miR-29c regulates RAG1 expression and modulates V(D)J recombination during B cell development.

Recombination activating genes (RAGs), consisting of RAG1 and RAG2, are stringently regulated lymphoid-specific genes, which initiate V(D)J recombination in developing lymphocytes. We report the regulation of RAG1 through a microRNA (miRNA), miR-29c, in a B cell stage-specific manner in mice and humans. Various lines of experimentation, including CRISPR-Cas9 genome editing, demonstrate the target specificity and direct interaction of miR-29c to RAG1. Modulation of miR-29c levels leads to change in V(D)J recombination efficiency in pre-B cells. The miR-29c expression is inversely proportional to RAG1 in a B cell developmental stage-specific manner, and miR-29c null mice exhibit a reduction in mature B cells. A negative correlation of miR-29c and RAG1 levels is also observed in leukemia patients, suggesting the potential use of miR-29c as a biomarker and a therapeutic target. Thus, our results reveal the role of miRNA in the regulation of RAG1 and its relevance in cancer.

Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis.

Host-virus protein-protein interactions play key roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity-labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Further characterization of these interactions and comparison to other large-scale study of cellular responses to SARS-CoV-2 infection elucidated how distinct SARS-CoV-2 viral proteins participate in its life cycle. With these data mining, we discovered potential drug targets for the treatment of COVID-19. The interactomes of two key SARS-CoV-2-encoded viral proteins, NSP1 and N, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of SARS-CoV-2 provides valuable resources for the understanding and treating of this disease.

Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2.

Poly (ADP-ribose) polymerase inhibitor (PARPi)-based therapies initially reduce tumor burden but eventually lead to acquired resistance in cancer patients with BRCA1 or BRCA2 mutation. To understand the potential PARPi resistance mechanisms, we performed whole-genome CRISPR screens to discover genetic alterations that change the gene essentiality in cells with inducible depletion of BRCA2. We identified that several RNA Polymerase II transcription Mediator complex components, especially Cyclin C (CCNC) as synthetic survival targets upon BRCA2 loss. Total mRNA sequencing demonstrated that loss of CCNC could activate the transforming growth factor (TGF)-beta signaling pathway and extracellular matrix (ECM)-receptor interaction pathway, however the inhibition of these pathways could not reverse cell survival in BRCA2 depleted CCNC-knockout cells, indicating that the activation of these pathways is not required for the resistance. Moreover, we showed that the improved survival is not due to restoration of homologous recombination repair although decreased DNA damage signaling was observed. Interestingly, loss of CCNC could restore replication fork stability in BRCA2 deficient cells, which may contribute to PARPi resistance. Taken together, our data reveal CCNC as a critical genetic determinant upon BRCA2 loss of function, which may help the development of novel therapeutic strategies that overcome PARPi resistance.

SCR7, an inhibitor of NHEJ can sensitize tumor cells to ionization radiation.

Nonhomologous end joining (NHEJ), one of the major DNA double-strand break repair pathways, plays a significant role in cancer cell proliferation and resistance to radio and chemotherapeutic agents. Previously, we had described a small molecule inhibitor, SCR7, which inhibited NHEJ in a DNA Ligase IV dependent manner. Here, we report that SCR7 potentiates the effect of γ-radiation (IR) that induces DNA breaks as intermediates to eradicate cancer cells. Dose fractionation studies revealed that coadministration of SCR7 and IR (0.5 Gy) in mice Dalton's lymphoma (DLA) model led to a significant reduction in mice tumor cell proliferation, which was equivalent to that observed for 2 Gy dose when both solid and liquid tumor models were used. Besides, co-treatment with SCR7 and 1 Gy of IR further improved the efficacy. Notably, there was no significant change in blood parameters, kidney and liver functions upon combinatorial treatment of SCR7 and IR. Further, the co-treatment of SCR7 and IR resulted in a significant increase in unrepaired DSBs within cancer cells compared to either of the agent alone. Anatomy, histology, and other studies in tumor models confirmed the cumulative effects of both agents in activating apoptotic pathways to induce cytotoxicity by modulating DNA damage response and repair pathways. Thus, we report that SCR7 has the potential to reduce the side effects of radiotherapy by lowering its effective dose ex vivo and in mice tumor models, with implications in cancer therapy.

Inhibition of the DNA damage response to activate immune responses toward tumors.

Cancer immunotherapy represents a very encouraging mode of treatment for cancer where one's immune system is utilized to eliminate tumor cells. Wayne et al. explore inhibition of DNA damage response (DDR) pathways with small molecule inhibitors as a means to prime cells with immune response. These findings suggest that a one-size-fits-all approach cannot be used when harnessing immune response via DDR inhibitors and genotoxic agents, which are required ultimately for the success of immunotherapy. Comment on: https://doi.org/10.1111/febs.15747.

C17orf53 is identified as a novel gene involved in inter-strand crosslink repair.

Ataxia Telangiectasia and Rad3-Related kinase (ATR) is a master regulator of genome maintenance, and participates in DNA replication and various DNA repair pathways. In a genome-wide screen for ATR-dependent fitness genes, we identified a previously uncharacterized gene, C17orf53, whose loss led to hypersensitivity to ATR inhibition. C17orf53 is conserved in vertebrates and is required for efficient cell proliferation. Loss of C17orf53 slowed down DNA replication and led to pronounced interstrand crosslink (ICL) repair defect. We showed that C17orf53 is a ssDNA- and RPA-binding protein and both characteristics are important for its functions in the cell. In addition, using multiple omics methods, we found that C17orf53 works with MCM8/9 to promote cell survival in response to ICL lesions. Taken together, our data suggest that C17orf53 is a novel component involved in ICL repair pathway.

FOXK1 Participates in DNA Damage Response by Controlling 53BP1 Function.

53BP1 plays a central role in dictating DNA repair choice between non-homologous end joining (NHEJ) and homologous recombination (HR), which is important for the sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPis) of BRCA1-deficient cancers. In this study, we show that FOXK1 associates with 53BP1 and regulates 53BP1-dependent functions. FOXK1-53BP1 interaction is significantly enhanced upon DNA damage during the S phase in an ATM/CHK2-dependent manner, which reduces the association of 53BP1 with its downstream factors RIF1 and PTIP. Depletion of FOXK1 impairs DNA repair and induces compromised cell survival upon DNA damage. Overexpression of FOXK1 diminishes 53BP1 foci formation, which leads to resistance to PARPis and elevation of HR in BRCA1-deficient cells and decreased telomere fusion in TRF2-depleted cells. Collectively, our findings demonstrate that FOXK1 negatively regulates 53BP1 function by inhibiting 53BP1 localization to sites of DNA damage, which alters the DSB-induced protein complexes centering on 53BP1 and thus influences DNA repair choice.

HMCES safeguards replication from oxidative stress and ensures error-free repair.

Replication across oxidative DNA lesions can give rise to mutations that pose a threat to genome integrity. How such lesions, which escape base excision repair, get removed without error during replication remains unknown. Our PCNA-based screen to uncover changes in replisome composition under different replication stress conditions had revealed a previously unknown PCNA-interacting protein, HMCES/C3orf37. Here, we show that HMCES is a critical component of the replication stress response, mainly upon base misincorporation. We further demonstrate that the absence of HMCES imparts resistance to pemetrexed treatment due to error-prone bypass of oxidative damage. Furthermore, based on genetic screening, we show that homologous recombination repair proteins, such as CtIP, BRCA2, BRCA1, and PALB2, are indispensable for the survival of HMCES KO cells. Hence, HMCES, which is the sole member of the SRAP superfamily in higher eukaryotes known so far, acts as a proofreader on replication forks, facilitates resolution of oxidative base damage, and therefore ensures faithful DNA replication.

AMPK Interactome Reveals New Function in Non-homologous End Joining DNA Repair.

Adenosine monophosphate-activated protein kinase (AMPK) is an obligate heterotrimer that consists of a catalytic subunit (α) and two regulatory subunits (ß and γ). AMPK is a key enzyme in the regulation of cellular energy homeostasis. It has been well studied and is known to function in many cellular pathways. However, the interactome of AMPK has not yet been systematically established, although protein-protein interaction is critically important for protein function and regulation. Here, we used tandem-affinity purification, coupled with mass spectrometry (TAP-MS) analysis, to determine the interactome of AMPK and its functions. We conducted a TAP-MS analysis of all seven AMPK subunits. We identified 138 candidate high-confidence interacting proteins (HCIPs) of AMPK, which allowed us to build an interaction network of AMPK complexes. Five candidate AMPK-binding proteins were experimentally validated, underlining the reliability of our data set. Furthermore, we demonstrated that AMPK acts with a strong AMPK-binding protein, Artemis, in non-homologous end joining. Collectively, our study established the first AMPK interactome and uncovered a new function of AMPK in DNA repair.

The ARK Assay Is a Sensitive and Versatile Method for the Global Detection of DNA-Protein Crosslinks.

DNA-protein crosslinks (DPCs) are a frequent form of DNA lesion and are strongly inhibitive in diverse DNA transactions. Despite recent developments, the biochemical detection of DPCs remains a limiting factor for the in-depth mechanistic understanding of DPC repair. Here, we develop a sensitive and versatile assay, designated ARK, for the quantitative analysis of DPCs in cells. ARK uses sequential chaotropic and detergent-based isolation of DPCs and substantially enhances sample purity, resulting in a 5-fold increase in detection sensitivity and a 10-fold reduction in background reading. We validate the ARK assay with genetic mutants with established deficiencies in DPC repair and demonstrate its robustness by using common DPC-inducing reagents, including formaldehyde, camptothecin, and etoposide. In addition, we show that the Fanconi anemia pathway contributes to the repair of DPCs. Thus, ARK is expected to facilitate various studies aimed at understanding both fundamental biology and translational applications of DNA-protein crosslink repair.

SLX4IP acts with SLX4 and XPF-ERCC1 to promote interstrand crosslink repair.

Interstrand crosslinks (ICLs) are highly toxic DNA lesions that are repaired via a complex process requiring the coordination of several DNA repair pathways. Defects in ICL repair result in Fanconi anemia, which is characterized by bone marrow failure, developmental abnormalities, and a high incidence of malignancies. SLX4, also known as FANCP, acts as a scaffold protein and coordinates multiple endonucleases that unhook ICLs, resolve homologous recombination intermediates, and perhaps remove unhooked ICLs. In this study, we explored the role of SLX4IP, a constitutive factor in the SLX4 complex, in ICL repair. We found that SLX4IP is a novel regulatory factor; its depletion sensitized cells to treatment with ICL-inducing agents and led to accumulation of cells in the G2/M phase. We further discovered that SLX4IP binds to SLX4 and XPF-ERCC1 simultaneously and that disruption of one interaction also disrupts the other. The binding of SLX4IP to both SLX4 and XPF-ERCC1 not only is vital for maintaining the stability of SLX4IP protein, but also promotes the interaction between SLX4 and XPF-ERCC1, especially after DNA damage. Collectively, these results demonstrate a new regulatory role for SLX4IP in maintaining an efficient SLX4-XPF-ERCC1 complex in ICL repair.

Role of Proton Pump Inhibitors in Laryngopharyngeal Reflux: Clinical Evaluation in a North Indian Population.

There has been an escalation of patients presenting with symptoms of Laryngopharyngeal reflux disease (LPRD) in the otorhinolaryngology clinics due to life style and dietary changes. This study was undertaken to evaluate the effect of various proton pump inhibitors in the treatment of LPRD using Reflux symptom index (RSI) and Reflux finding score (RFS). This was a prospective study conducted from June 2016 to February 2017 with a total of 240 patients with symptoms and signs of LPR. The patients were divided into 5 groups. Each group was subjected to particular proton pump inhibitor. There were 124 males 116 females with a mean age 34.3 and rural to urban ratio being 11. After 3 months, RFS and RSI score within each group, improved significantly with Proton pump inhibitor therapy. In our study patients who were treated with omeprazole 20 mg twice daily had the highest improvement in laryngeal symptoms and laryngeal findings. We conclude emphasizing the effectiveness of proton pump inhibitors with incorporation of lifestyle modification in the successful management of LPRD.

Global phosphoproteomic analysis reveals ARMC10 as an AMPK substrate that regulates mitochondrial dynamics.

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis. Although AMPK has been studied extensively in cellular processes, understanding of its substrates and downstream functional network, and their contributions to cell fate and disease development, remains incomplete. To elucidate the AMPK-dependent signaling pathways, we performed global quantitative phosphoproteomic analysis using wild-type and AMPKα1/α2-double knockout cells and discovered 160 AMPK-dependent phosphorylation sites. Further analysis using an AMPK consensus phosphorylation motif indicated that 32 of these sites are likely direct AMPK phosphorylation sites. We validated one uncharacterized protein, ARMC10, and demonstrated that the S45 site of ARMC10 can be phosphorylated by AMPK both in vitro and in vivo. Moreover, ARMC10 overexpression was sufficient to promote mitochondrial fission, whereas ARMC10 knockout prevented AMPK-mediated mitochondrial fission. These results demonstrate that ARMC10 is an effector of AMPK that participates in dynamic regulation of mitochondrial fission and fusion.

Genome-wide CRISPR screens reveal synthetic lethality of RNASEH2 deficiency and ATR inhibition.

Ataxia telangiectasia mutated and RAD3 related (ATR) protein kinase plays critical roles in ensuring DNA replication, DNA repair, and cell cycle control in response to replication stress, making ATR inhibition a promising therapeutic strategy for cancer treatment. To identify genes whose loss makes tumor cells hypersensitive to ATR inhibition, we performed CRISPR/Cas9-based whole-genome screens in 3 independent cell lines treated with a highly selective ATR inhibitor, AZD6738. These screens uncovered a comprehensive genome-wide profile of ATR inhibitor sensitivity. From the candidate genes, we demonstrated that RNASEH2 deficiency is synthetic lethal with ATR inhibition both in vitro and in vivo. RNASEH2-deficient cells exhibited elevated levels of DNA damage and, when treated with AZD6738, underwent apoptosis (short-time treated) or senescence (long-time treated). Notably, RNASEH2 deficiency is frequently found in prostate adenocarcinoma; we found decreased RNASEH2B protein levels in prostate adenocarcinoma patient-derived xenograft (PDX) samples. Our findings suggest that ATR inhibition may be beneficial for cancer patients with reduced levels of RNASEH2 and that RNASEH2 merits further exploration as a potential biomarker for ATR inhibitor-based therapy.

Replisome Dynamics and Their Functional Relevance upon DNA Damage through the PCNA Interactome.

Eukaryotic cells use copious measures to ensure accurate duplication of the genome. Various genotoxic agents pose threats to the ongoing replication fork that, if not efficiently dealt with, can result in replication fork collapse. It is unknown how replication fork is precisely controlled and regulated under different conditions. Here, we examined the complexity of replication fork composition upon DNA damage by using a PCNA-based proteomic screen to uncover known and unexplored players involved in replication and replication stress response. We used camptothecin or UV radiation, which lead to fork-blocking lesions, to establish a comprehensive proteomics map of the replisome under such replication stress conditions. We identified and examined two potential candidate proteins WIZ and SALL1 for their roles in DNA replication and replication stress response. In addition, our unbiased screen uncovered many prospective candidate proteins that help fill the knowledge gap in understanding chromosomal DNA replication and DNA repair.

Deregulation of CRAD-controlled cytoskeleton initiates mucinous colorectal cancer via ß-catenin.

Epithelial integrity is maintained by the cytoskeleton and through cell adhesion. However, it is not yet known how a deregulated cytoskeleton is associated with cancer. We identified cancer-related regulator of actin dynamics (CRAD) as frequently mutated or transcriptionally downregulated in colorectal cancer. We found that CRAD stabilizes the cadherin-catenin-actin complex via capping protein inhibition. The loss of CRAD inhibits F-actin polymerization and subsequently disrupts the cadherin-catenin-actin complex, which leads to ß-catenin release and Wnt signalling hyperactivation. In mice, CRAD knockout induces epithelial cell integrity loss and Wnt signalling activation, resulting in the development of intestinal mucinous adenoma. With APC mutation, CRAD knockout initiates and accelerates mucinous and invasive adenoma development in the colorectum. These results define CRAD as a tumour suppressor, the inactivation of which deregulates the cytoskeleton and hyperactivates Wnt signalling thus initiating mucinous colorectal cancer. Our study reveals the unexpected roles of an actin cytoskeletal regulator in maintaining epithelial cell integrity and suppressing tumorigenesis.