Bifunctional Tumor-Targeted Bioprobe for Phothotheranosis.

Background: Near-infrared (NIR) phototheranostics provide promising noninvasive imaging and treatment for head and neck squamous cell carcinoma (HNSCC), capitalizing on its adjacency to skin or mucosal surfaces. Activated by laser irradiation, targeted NIR fluorophores can selectively eradicate cancer cells, harnessing the power of synergistic photodynamic therapy and photothermal therapy. However, there is a paucity of NIR bioprobes showing tumor-specific targeting and effective phototheranosis without hurting surrounding healthy tissues. Methods: We engineered a tumor-specific bifunctional NIR bioprobe designed to precisely target HNSCC and induce phototheranosis using bioconjugation of a cyclic arginine-glycine-aspartic acid (cRGD) motif and zwitterionic polymethine NIR fluorophore. The cytotoxic effects of cRGD-ZW800-PEG were measured by assessing heat and reactive oxygen species (ROS) generation upon an 808-nm laser irradiation. We then determined the in vivo efficacy of cRGD-ZW800-PEG in the FaDu xenograft mouse model of HNSCC, as well as its biodistribution and clearance, using a customized portable NIR imaging system. Results: Real-time NIR imaging revealed that intravenously administered cRGD-ZW800-PEG targeted tumors rapidly within 4 h postintravenous injection in tumor-bearing mice. Upon laser irradiation, cRGD-ZW800-PEG produced ROS and heat simultaneously and exhibited synergistic photothermal and photodynamic effects on the tumoral tissue without affecting the neighboring healthy tissues. Importantly, all unbound bioprobes were cleared through renal excretion. Conclusions: By harnessing phototheranosis in combination with tailored tumor selectivity, our targeted bioprobe ushers in a promising paradigm in cancer treatment. It promises safer and more efficacious therapeutic avenues against cancer, marking a substantial advancement in the field.

ssDNA reeling is an intermediate step in the reiterative DNA unwinding activity of the WRN-1 helicase.

RecQ helicases are highly conserved between bacteria and humans. These helicases unwind various DNA structures in the 3' to 5'. Defective helicase activity elevates genomic instability and is associated with predisposition to cancer and/or premature aging. Recent single-molecule analyses have revealed the repetitive unwinding behavior of RecQ helicases from Escherichia coli to humans. However, the detailed mechanisms underlying this behavior are unclear. Here, we performed single-molecule studies of WRN-1 Caenorhabditis elegans RecQ helicase on various DNA constructs and characterized WRN-1 unwinding dynamics. We showed that WRN-1 persistently repeated cycles of DNA unwinding and rewinding with an unwinding limit of 25 to 31 bp per cycle. Furthermore, by monitoring the ends of the displaced strand during DNA unwinding we demonstrated that WRN-1 reels in the ssDNA overhang in an ATP-dependent manner. While WRN-1 reeling activity was inhibited by a C. elegans homolog of human replication protein A, we found that C. elegans replication protein A actually switched the reiterative unwinding activity of WRN-1 to unidirectional unwinding. These results reveal that reeling-in ssDNA is an intermediate step in the reiterative unwinding process for WRN-1 (i.e., the process proceeds via unwinding-reeling-rewinding). We propose that the reiterative unwinding activity of WRN-1 may prevent extensive unwinding, allow time for partner proteins to assemble on the active region, and permit additional modulation in vivo.

DNA polymerase θ-mediated repair of high LET radiation-induced complex DNA double-strand breaks.

DNA polymerase θ (POLQ) is a unique DNA polymerase that is able to perform microhomology-mediated end-joining as well as translesion synthesis (TLS) across an abasic (AP) site and thymine glycol (Tg). However, the biological significance of the TLS activity is currently unknown. Herein we provide evidence that the TLS activity of POLQ plays a critical role in repairing complex DNA double-strand breaks (DSBs) induced by high linear energy transfer (LET) radiation. Radiotherapy with high LET radiation such as carbon ions leads to more deleterious biological effects than corresponding doses of low LET radiation such as X-rays. High LET-induced DSBs are considered to be complex, carrying additional DNA damage such as AP site and Tg in close proximity to the DSB sites. However, it is not clearly understood how complex DSBs are processed in mammalian cells. We demonstrated that genetic disruption of POLQ results in an increase of chromatid breaks and enhanced cellular sensitivity following treatment with high LET radiation. At the biochemical level, POLQ was able to bypass an AP site and Tg during end-joining and was able to anneal two single-stranded DNA tails when DNA lesions were located outside the microhomology. This study offers evidence that POLQ is directly involved in the repair of complex DSBs.

Whole Mitochondrial Genome Analysis in Non-Small Cell Lung Carcinoma Reveals Unique Tumor-Specific Somatic Mutations.

CONTEXT.­: Mitochondria and mitochondrial DNA have been suggested to play a role in cancer initiation and progression. Knowledge of mitochondrial DNA could provide a breakthrough to advance cancer management. OBJECTIVE.­: To identify the mitochondrial DNA landscape in non-small cell lung carcinoma. DESIGN.­: The adenocarcinoma set consisted of 365 pairs of adenocarcinomas and normal lung tissues, whereas the metastasis set included 12 primary non-small cell carcinomas, 15 metastatic tumors, and their normal counterparts. Tumor-specific somatic variants were identified, and if a variant showed heteroplasmy, the proportion of minor alleles was evaluated. Variants with greater than 10% change in allele frequency between tumor and normal pairs were identified as "heteroplasmic shifts." RESULTS.­: Tumor-specific variants appeared throughout the whole mitochondrial genome, without a common hot spot. Distinct variant profiles were seen in 289 (79.18%) of all individual adenocarcinomas. The presence of a unique profile and the number and loading of heteroplasmic shifts in tumors increased with higher stage or lymph node metastasis, and were related to shorter survival. In the metastasis set, the primary tumor variants were generally found in metastatic tumors. CONCLUSIONS.­: This study shows that somatic mitochondrial DNA mutations present with diverse locations and unique profiles in each individual tumor, implying their clinicopathologic utility.

HSA-ZW800-PEG for Enhanced Optophysical Stability and Tumor Targeting.

Small molecule fluorophores often face challenges such as short blood half-life, limited physicochemical and optical stability, and poor pharmacokinetics. To overcome these limitations, we conjugated the zwitterionic near-infrared fluorophore ZW800-PEG to human serum albumin (HSA), creating HSA-ZW800-PEG. This conjugation notably improves chemical, physical, and optical stability under physiological conditions, addressing issues commonly encountered with small molecules in biological applications. Additionally, the high molecular weight and extinction coefficient of HSA-ZW800-PEG enhances biodistribution and tumor targeting through the enhanced permeability and retention effect. The unique distribution and elimination dynamics, along with the significantly extended blood half-life of HSA-ZW800-PEG, contribute to improved tumor targetability in both subcutaneous and orthotopic xenograft tumor-bearing animal models. This modification not only influences the pharmacokinetic profile, affecting retention time and clearance patterns, but also enhances bioavailability for targeting tissues. Our study guides further development and optimization of targeted imaging agents and drug-delivery systems.

The dynamic landscape of transcription initiation in yeast mitochondria.

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.

Tonicity-responsive enhancer-binding protein promotes stemness of liver cancer and cisplatin resistance.

BACKGROUND: High recurrence and chemoresistance drive the high mortality in hepatocellular carcinoma (HCC). Although cancer stem cells are considered to be the source of recurrent and chemoresistant tumors, they remain poorly defined in HCC. Tonicity-responsive enhancer binding protein (TonEBP) is elevated in almost all HCC tumors and associated with recurrence and death. We aimed to identify function of TonEBP in stemness and chemoresistance of liver cancer. METHODS: Tumors obtained from 280 HCC patients were analyzed by immunohistochemical analyses. Stemness and chemoresistance of liver CSCs (LCSCs) were investigated using cell culture. Tumor-initiating activity was measured by implanting LCSCs into BALB/c nude mice. FINDINGS: Expression of TonEBP is higher in LCSCs in HCC cell lines and correlated with markers of LCSCs whose expression is significantly associated with poor prognosis of HCC patients. TonEBP mediates ATM-mediated activation of NF-κB, which stimulates the promoter of a key stem cell transcription factor SOX2. As expected, TonEBP is required for the tumorigenesis and self-renewal of LSCSs. Cisplatin induces the recruitment of the ERCC1/XPF dimer to the chromatin in a TonEBP-dependent manner leading to DNA repair and cisplatin resistance. The cisplatin-induced inflammation in LSCSs is also dependent on the TonEBP-ERCC1/XPF complex, and leads to enhanced stemness via the ATM-NF-κB-SOX2 pathway. In HCC patients, tumor expression of ERCC1/XPF predicts recurrence and death in a TonEBP-dependent manner. INTERPRETATION: TonEBP promotes stemness and cisplatin resistance of HCC via ATM-NF-κB. TonEBP is a key regulator of LCSCs and a promising therapeutic target for HCC and its recurrence.

Comparison of Bone Regeneration between Porcine-Derived and Bovine-Derived Xenografts in Rat Calvarial Defects: A Non-Inferiority Study.

The present study aimed to compare the bone-regeneration capacity of porcine-derived xenografts to bovine-derived xenografts in the rat calvarial defect model. The observation of surface morphology and in vitro cell studies were conducted prior to the animal study. Defects with a diameter of 8 mm were created in calvaria of 20 rats. The rats were randomly treated with porcine-derived (Bone-XP group) or bovine-derived xenografts (Bio-Oss group) and sacrificed at 4 and 8 weeks after surgery. The new bone regeneration was evaluated by micro-computed tomography (µCT) and histomorphometric analyses. In the cell study, the extracts of Bone-XP and Bio-Oss showed a positive effect on the regulation of osteogenic differentiation of human mesenchymal stem cells (hMSCs) without cytotoxicity. The new bone volume of Bone-XP (17.52 ± 3.78% at 4 weeks and 32.09 ± 3.51% at 8 weeks) was similar to that of Bio-Oss (11.6 ± 3.88% at 4 weeks and 25.89 ± 7.43% at 8 weeks) (p > 0.05). In the results of new bone area, there was no significant difference between Bone-XP (9.08 ± 5.47% at 4 weeks and 25.22 ± 13.56% at 8 weeks) and Bio-Oss groups (5.83 ± 2.56% at 4 weeks and 21.68 ± 11.11% at 8 weeks) (p > 0.05). It can be concluded that the porcine-derived bone substitute may offer a favorable cell response and bone regeneration similar to those of commercial bovine bone mineral.

Molecular characteristics of reiterative DNA unwinding by the Caenorhabditis elegans RecQ helicase.

The RecQ family of helicases is highly conserved both structurally and functionally from bacteria to humans. Defects in human RecQ helicases are associated with genetic diseases that are characterized by cancer predisposition and/or premature aging. RecQ proteins exhibit 3'-5' helicase activity and play critical roles in genome maintenance. Recent advances in single-molecule techniques have revealed the reiterative unwinding behavior of RecQ helicases. However, the molecular mechanisms involved in this process remain unclear, with contradicting reports. Here, we characterized the unwinding dynamics of the Caenorhabditis elegans RecQ helicase HIM-6 using single-molecule fluorescence resonance energy transfer measurements. We found that HIM-6 exhibits reiterative DNA unwinding and the length of DNA unwound by the helicase is sharply defined at 25-31 bp. Experiments using various DNA substrates revealed that HIM-6 utilizes the mode of 'sliding back' on the translocated strand, without strand-switching for rewinding. Furthermore, we found that Caenorhabditis elegans replication protein A, a single-stranded DNA binding protein, suppresses the reiterative behavior of HIM-6 and induces unidirectional, processive unwinding, possibly through a direct interaction between the proteins. Our findings shed new light on the mechanism of DNA unwinding by RecQ family helicases and their co-operation with RPA in processing DNA.

Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes.

Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. Here, we show the distinct PCNA loading or unloading activity of each clamp loader. ATAD5-RLC possesses the potent PCNA unloading activity. ATPase motif and collar domain of ATAD5 are crucial for the unloading activity. DNA structures did not affect PCNA unloading activity of ATAD5-RLC. ATAD5-RLC could unload ubiquitinated PCNA. Through single molecule measurements, we reveal that ATAD5-RLC unloaded PCNA through one intermediate state before ATP hydrolysis. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis. Replication proteins such as Fen1 could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Our findings provide molecular insights into how PCNA is released from chromatin to finalize DNA replication/repair.

Opening-closing dynamics of the mitochondrial transcription pre-initiation complex.

Promoter recognition and local melting of DNA are key steps of transcription initiation catalyzed by RNA polymerase and initiation factors. From single molecule fluorescence resonance energy transfer studies of the yeast (Saccharomyces cerevisiae) mitochondrial RNA polymerase Rpo41 and its transcription factor Mtf1, we show that the pre-initiation complex is highly dynamic and undergoes repetitive opening-closing transitions that are modulated by Mtf1 and ATP. We found that Rpo41 alone has the intrinsic ability to bend the promoter but only very briefly. Mtf1 enhances bending/opening transition and suppresses closing transition, indicating its dual roles of nucleating promoter opening and stabilizing the open state. The cognate initiating ATP prolongs the lifetime of the open state, plausibly explaining the 'ATP sensing mechanism' suggested for the system. We discovered short-lived opening trials upon initial binding of Rpo41-Mtf1 before the establishment of the opening/closing equilibrium, which may aid in promoter selection before the formation of stable pre-initiation complex. The dynamics of open complex formation provides unique insights into the interplay between RNA polymerase and transcription factors in regulating initiation.

Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity.

Single-molecule FRET has been widely used for monitoring protein-nucleic acids interactions. Direct visualization of the interactions, however, often requires a site-specific labeling of the protein, which can be circuitous and inefficient. In addition, FRET is insensitive to distance changes in the 0-3-nm range. Here, we report a systematic calibration of a single molecule fluorescence assay termed protein induced fluorescence enhancement. This method circumvents protein labeling and displays a marked distance dependence below the 4-nm distance range. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein. Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. We demonstrate the general applicability and distance sensitivity using (a) a finely spaced DNA ladder carrying a restriction site for BamHI, (b) RNA translocation by DExH enzyme RIG-I, and (c) filament dynamics of RecA on single-stranded DNA. The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.