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The antenna proposed in this article is structured on a Rogers RT5880 (lossy) substrate holding two grounds parallel on two opposite planes, one adjacent to the transmission path feeding the fractal patch. These grounds are named as parallel−plane grounds (PPGs). The fractal antenna features a compact size of 17.7×9.8×1.575 mm3 unveiling a −10 dB impedance bandwidth of 19.5 GHz (3.2 GHz–22.7 GHz). The iterative ratio, r has an impact on the impedance bandwidth resembling other design parameters. A transient bandwidth enhancement is achievable by keeping the iterative ratio in a range of (0.6–1.1). In this range, the bandwidth gets enhanced as r ascends. The ground adjacent to the feeding path helped to achieve responses in X band (8 GHz–12 GHz), and Ku band (12 GHz–18 GHz) crossing the range of the whole ultra−wideband (3.1 GHz–10.6 GHz). It responds in some spectrum (18 GHz–22.7 GHz) of the K band (18 GHz–27 GHz) also.

Summary: This article presents a highly compact printed radiator for direct broadcast satellite (DBS) systems that exhibits good gain and left‐handed circular polarization. The inferred pattern is accomplished by etching two isosceles triangles, one from each of the four corners of a square. From the center of the newly acquired structure, a circular geometric design is made up of 16 isosceles‐scaled triangles has been etched. This arrangement induces resonance in the antenna, aligning it with the desired frequency. The proposed antenna is designed using PCB laminated Rogger 5880 material and simulated in a CST EM simulator. The antenna under consideration exhibits a significant size reduction, achieving a miniaturization ratio of 21.03%. The LHCP antenna resonated at 12.61 GHz and offered a decent LHCP gain of 7.95 dBic. It gives 0.56 GHz CP bandwidth, which covers the DBS service band entirely. It also provides 96.43% radiation efficiency and 44.27 dB/m correction factor, respectively. All the measured findings of the prototype agree with its simulated one. [ABSTRACT FROM AUTHOR]

Cell-free protein synthesis (CFPS) is a method utilized for producing proteins without the limits of cell viability. The plug-and-play utility of CFPS is a key advantage over traditional plasmid-based expression systems and is foundational to the potential of this biotechnology. A key limitation of CFPS is the varying stability of DNA types, limiting the effectiveness of cell-free protein synthesis reactions. Researchers generally rely on plasmid DNA for its ability to support robust protein expression in vitro. However, the overhead required to clone, propagate, and purify plasmids reduces the potential of CFPS for rapid prototyping. While linear templates overcome the limits of plasmid DNA preparation, linear expression templates (LETs) were under-utilized due to their rapid degradation in extract based CFPS systems, limiting protein synthesis. To reach the potential of CFPS using LETs, researchers have made notable progress toward protection and stabilization of linear templates throughout the reaction. The current advancements range from modular solutions, such as supplementing nuclease inhibitors and genome engineering to produce strains lacking nuclease activity. Effective application of LET protection techniques improves expression yields of target proteins to match that of plasmid-based expression. The outcome of LET utilization in CFPS is rapid design–build–test–learn cycles to support synthetic biology applications. This review describes the various protection mechanisms for linear expression templates, methodological insights for implementation, and proposals for continued efforts that may further advance the field. [ABSTRACT FROM AUTHOR]

The Ku heterodimer acts centrally in nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB). Saccharomyces cerevisiae Ku, like mammalian Ku, binds and recruits NHEJ factors to DSB ends. Consequently, NHEJ is virtually absent in yeast Ku null (yku70∆ or yku80∆) strains. Previously, we unexpectedly observed imprecise NHEJ proficiency in a yeast Ku mutant with impaired DNA end-binding (DEB). However, how DEB impairment supported imprecise NHEJ was unknown. Here, we found imprecise NHEJ proficiency to be a feature of a panel of DEB-impaired Ku mutants and that DEB impairment resulted in a deficiency in precise NHEJ. These results suggest that DEB-impaired Ku specifically promotes error-prone NHEJ. Epistasis analysis showed that classical NHEJ factors, as well as novel and previously characterized NHEJ-specific residues of Ku, are required for the distinct error-prone repair in a Ku DEB mutant. However, sequencing of repair junctions revealed that imprecise repair in Ku DEB mutants was almost exclusively characterized by small deletions, in contrast to the majority of insertions that define imprecise repair in wild-type strains. Notably, while sequencing indicated a lack of Pol4-dependent insertions at the site of repair, Pol2 exonuclease activity, which mediates small deletions in NHEJ, contributed to imprecise NHEJ in a Ku DEB mutant. The deletions were smaller than in Ku-independent microhomology-mediated end-joining (MMEJ) and were neither promoted by Mre11 nuclease activity nor Sae2 Thus, the quality of Kus engagement at the DNA end influences end-processing during NHEJ and DEB impairment unmasks a Ku-dependent error-prone pathway of end-joining distinct from MMEJ.

DNA-dependent protein kinase (DNA-PK) is a key player in the NHEJ repair pathway. DNA-PK and its subunits, Ku70, Ku80, and catalytic subunit (DNA-PKcs), also participate in other cellular processes; however, there are still no systemic data on the effect of depletion of Ku70, Ku80 and DNA-PKcs on cell functions in the same cell line. Here, we analyzed transcriptome changes in HEK 293T cells after depletion of each DNA-PK subunit. Depletion of various DNA-PK subunits resulted in dramatic differences in the number of differentially expressed genes: only 7 genes changed more than 2-fold in DNA-PKcs-deficient cells, 29 genes in Ku80-deficient, 219 genes in Ku70-deficient. All DNA-PKcs-dependent genes were stress-related and depended on both Ku70 and Ku80. Two-thirds of Ku80-dependent genes were also differentially expressed in the Ku70-deficient line. Most Ku70-dependent genes were altered exclusively in Ku70-depleted cells, indicating that Ku70 is involved in the regulation of more processes than Ku80. GO enrichment analysis showed the effect of Ku70 knockdown on cell adhesion and matrix organization, protein degradation, cell proliferation, and differentiation. Depletion of Ku70, but not Ku80, provided greater cell motility and disassembly of cell-cell contacts. These data clearly indicate that Ku70 is more functionally important for the cell life than DNA-PKcs and even Ku80. • Transcriptome analysis of cells with depletion of each of the DNA-PK subunits is done. • Depletion of Ku70 changes expression of more genes than depletion of Ku80. • Depletion of Ku70 increases cell motility and disassembly of cell-cell contacts. [ABSTRACT FROM AUTHOR]

Background: Differentiating Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is very important in the treatment process of patients. According to the American Thoracic Society guideline (ATS), NTM clinical isolates should be identified at the species level proper treatment and patient management. This study aimed to identify NTM clinical isolates by evaluationg rpoB, ssrA, tuf, atpE, ku, and dnaK genes, and use multilocus sequence analysis (MLSA) to concatenate the six genes. Methods: Ninety-six Mycobacterium isolates, including 86 NTM and 10 MTB isolates, from all the patients referred to the certain TB Reference Centres were included. All isolates were evaluated by PCR amplification of rpoB, ssrA, tuf, ku, atpE, and dnaK genes and MLSA. Results: Out of 96 isolates, 91 (94.8%), 87 (90.6%), 72 (75%), 84 (87.5%) and 79 (82.3%) were differentiated to the species level by rpoB, tuf, ssrA, dnaK and atpE genes, respectively. The ku gene was able to identify 69 (80.2%) isolates of the 86 NTM isolates to the species level. We could identify 100% of the isolates to the species level by MLSA. Conclusions: None of the PCR targets used in this study were able to completely differentiate all species. The MLSA technique used to concatenate the six genes could increase the identification of clinical Mycobacterium isolates and all 16 species were well-differentiated. [ABSTRACT FROM AUTHOR]

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.

A continuous beam-steering solution for circularly polarized (CP) radial-line slot array (RLSA) antennas is presented and demonstrated with a fabricated prototype. The solution is based on the near-field meta-steering (NFMS) method, which is implemented through a pair of highly transmitting hybrid metasurfaces (HMs) that are placed above a high-gain CP RLSA antenna in the near-field region. Cells in HMs, unlike conventional metasurfaces (CMs), can provide exact or close to exact phase shift with transmission magnitude larger than −1 dB. This is achieved by combining the nonoverlapping phase ranges of two dramatically different phase-shifting cells. The RLSA is stationary, while the two HMs are rotated to steer the beam in a 2-D (azimuth and elevation) space. The measured results of the prototype demonstrate that the system can steer its beam to a maximum elevation angle of 40.6° with a maximum gain of 30.9 dBic. The axial ratio remained less than 3 dB in the 3 dB beamwidth even when the beam is steered. The height of the system is only 4.5 cm, which is much less than mechanically steered reflector antennas. Unlike electronically steered high-gain antenna arrays, this system requires only few watts of power only when rotating HMs and has zero power consumption when the beam is stationary. [ABSTRACT FROM AUTHOR]

Objectives: Corynebacterium glutamicum (C. glutamicum) has been harnessed for multi-million-ton scale production of glutamate and lysine. To further increase its amino acid production for fermentation industry, there is an acute need to develop next-generation genome manipulation tool for its metabolic engineering. All reported methods for genome editing triggered with CRISPR-Cas are based on the homologous recombination. While, it requires the generation of DNA repair template, which is a bottle-neck for its extensive application. Results: In this study, we developed a method for gene knockout in C. glutamicum via CRISPR-Cpf1-coupled non-homologous end-joining (CC-NHEJ). Specifically, CRISPR-Cpf1 introduced double-strand breaks in the genome of C. glutamicum, which was further repaired by ectopically expressed two NHEJ key proteins (Mycobacterium tuberculosis Ku and ligase D). We provide the proof of concept, for CC-NHEJ, by the successful knockout of the crtYf/e gene in C. glutamicum with the efficiency of 22.00 ± 5.56%, or something like that. Conclusion: The present study reported a novel genome manipulation method for C. glutamicum. [ABSTRACT FROM AUTHOR]

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role. • Mutation of the KBM-binding domain in KU80 increases sensitivity to DNA replication stress • CHO cells expressing KU80 with a mutated KBM-binding domain accumulate high levels of pan-nuclear γH2AX in S/G2 phase • This phenotype is also observed in CHO cells in which the KBM-containing protein WRN is depleted. • These data implicate the KBM-binding domain in KU80 in the response to replication stress, and suggest that this role is facilitated via WRN. [ABSTRACT FROM AUTHOR]

Ionizing radiation exerts various biological effects, including cell killing and carcinogenesis, mainly through generating damage on DNA. Among various types of DNA damage, DNA double-strand break (DSB) is considered the most deleterious and most intimately related to biolog?ical effects of radiation. DNA-dependent protein kinase (DNA-PK), consisting of DNA-PK catalytic subunit and Ku80-Ku70 heterodimer (Ku), is activated upon binding to the end of double-stranded DNA and acts as the molecular sensor for DSB. While DSB is repaired mainly through homologous recombination and nonhomologous end joining in eukaryotes, DNA-PK is shown to be essential in the latter pathway. Moreover, DNA-PK is reported to be capable of phosphorylating a number of proteins, suggesting versatile functions of DNA-PK in cellular response to DSB. Here, we review the advance in our understanding on DNA-PK in three decades and remaining problems.

Studies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.

The broadband, narrow width, rectangular dielectric resonator antenna (RDRA) of aluminum nitride (εr=8.6) was designed and the effect of inclusion of air gap at the bottom of the dielectric resonator antenna (DRA), above the ground plane, was investigated. Gain around 7 dBi was obtained for DRA with air gap (DRAAG) over a broad bandwidth in upper X, Ku, and K bands. Further enhancement in gain could be obtained by placing a metal wall parallel to the length of DRA. However, due to the presence of metal wall, bandwidth was reduced. These structures with the metal wall are capable of operating over a wide band extending from Ku band to lower K band with the gain of around 10 dBi. CST Microwave Studio Software was used to simulate all these structures. Performance parameters of DRA with air gap were compared with several broadband DRA structures reported in recent literature. The proposed DRAAG with the metal wall in this paper is capable of operating over a wide bandwidth along with a significant gain.

Airway epithelial homeostasis is under constant threat due to continuous exposure to the external environment, and abnormally robust sensitivity to external stimuli is critical to the development of airway diseases, including asthma. Ku is a key nonhomologous end-joining DNA repair protein with diverse cellular functions such as VDJ recombination and telomere length maintenance. Here, we show a novel function of Ku in alleviating features of allergic airway inflammation via the regulation of mitochondrial and endoplasmic reticulum (ER) stress. We first determined that airway epithelial cells derived from both asthmatic lungs and murine asthma models demonstrate increased expression of 8-hydroxy-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage. Ku protein expression was dramatically reduced in the bronchial epithelium of patients with asthma as well as in human bronchial epithelial cells exposed to oxidative stress. Knockdown of Ku70 or Ku80 in naïve mice elicited mitochondrial collapse or ER stress, leading to bronchial epithelial cell apoptosis and spontaneous development of asthma-like features, including airway hyperresponsiveness, airway inflammation, and subepithelial fibrosis. These findings demonstrate an essential noncanonical role for Ku proteins in asthma pathogenesis, likely via maintenance of organelle homeostasis. This novel function of Ku proteins may also be important in other disease processes associated with organelle stress. [ABSTRACT FROM AUTHOR]

DNA double‐strand breaks (DSBs) are one of the most serious types of DNA damage. However, multiple repair pathways are present in cells to ensure rapid and appropriate repair of DSBs. Pathway selection depends on several factors including cell type, cell cycle phase, and damage severity. Ribosomal protein S3 (rpS3), a component of the 40S small ribosomal subunit, is a multi‐functional protein primarily involved in protein synthesis. rpS3 is also involved in the mediation of various extra‐ribosomal pathways, including DNA damage processing and the stress response. Here, we report that rpS3 is a novel negative regulator of non‐homologous end joining (NHEJ)‐mediated repair of DSBs. We found that rpS3 interacts with the Ku heterodimers of the DNA‐dependent protein kinase (DNA‐PK) complex and slows down NHEJ ligation reactions, ultimately triggering p53‐dependent cell death following treatment with high‐dose ionizing radiation. After DSB formation, DNA‐PK phosphorylates rpS3, which consequently reduces the binding of rpS3 to the Ku complex. We hypothesized that rpS3 may play a role in DSB repair by repressing NHEJ, while inducing other repair pathways, and by initiating DSB‐induced cell death in response to severe DNA damage. [ABSTRACT FROM AUTHOR]

In vertebrates, double-strand breaks in DNA are primarily repaired by Non-Homologous End-Joining (NHEJ). The ring-shaped Ku heterodimer rapidly senses and threads onto broken DNA ends forming a recruiting hub. Through protein-protein contacts eventually reinforced by protein-DNA interactions, the Ku-DNA hub attracts a series of specialized proteins with scaffolding and/or enzymatic properties. To shed light on these dynamic interplays, we review here current knowledge on proteins directly interacting with Ku and on the contact points involved, with a particular accent on the different classes of Ku-binding motifs identified in several Ku partners. An integrated structural model of the core NHEJ network at the synapsis step is proposed. [ABSTRACT FROM AUTHOR]

In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a c-NHEJ core component. We report here that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(D)J recombination assays, and do not shift toward higher micro-homology usage in plasmid repair assays. Although PAXX-deficient cells lack c-NHEJ phenotypes, PAXX forms a stable ternary complex with Ku bound to DNA. Formation of this complex involves an interaction with Ku70 and requires a bare DNA extension for stability. Moreover, the relatively weak Ku-dependent stimulation of LIG4/XRCC4 activity by PAXX is unmasked by XLF ablation. Thus, PAXX plays an accessory role during c-NHEJ that is largely overlapped by XLF’s function.

Repair of DNA double strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Given its high affinity for ends, multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ~1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unknown. Here we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process which actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism which operates in S-phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together our data shed light on the multiple layers of coordinated mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions., Competing Interests: Declaration of interests. The authors declare no competing interests.

Here we report that BCL2 blocks DNA double strand break (DSB) repair via nonhomologous end-joining (NHEJ), through sequestration of KU80 protein outside the nucleus. We find that this effect is associated with a repair switch to the error-prone PARP1-dependent end-joining (PARP1-EJ). We present in-vitro proof-of-concept for therapeutic targeting of this switch using PARP inhibitor to specifically enhance the radiosensitivity of BCL2-overexpressing cells. Given its erroneous behavior, PARP1-EJ might allow for the accumulation of genetic alterations and tumor progression. Consistently, we report an inverse correlation between BCL2 expression and biochemical recurrence-free survival of 10.259 prostate cancer (PCa) patients who underwent primary radical-prostatectomy for localized disease. Further, we evaluated retrospectively the impact of BCL2 expression on clinical outcome of 1.426 PCa patients, who had been given salvage radiotherapy at relapse after radical prostatectomy. In line with its role in blocking NHEJ, BCL2 over-expressers showed significantly better response to salvage radiotherapy compared to low-expressers. Collectively, our findings identify BCL2 status in PCa as a putative predictor of (i) radiotherapy response and (ii) response to treatment with PARP inhibitor olaparib as a radiosensitizing agent. [ABSTRACT FROM AUTHOR]

Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25 bp dsDNA or 25 bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35 bp blunt ended dsDNA) or 25 bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes. [ABSTRACT FROM AUTHOR]

Attenuation due to rain predictive models have been used to calculate the impact of rainfall on satellite communication for six stations in Malaysia. The impact of rainfall is very important for designing a modern satellite system for heavy rainfall climatic regions like Malaysia, with large annual rainfall accumulation exceeding 3000 mm and rainfall rate exceeding 150 mm/h at Ku (12/14 GHz), Ka (20/30 GHz) and V (40/50 GHz) bands. The present result shows that the avalibility of link for 99.99% at the three bands for uplink and downlink to Malaysian Communication Satellite (MEASAT-3a) is not practicable. The results suggest link availability of 99.9% for Ku-uplink and Ka downlink, while 99% for Ka uplink and 99% for V band uplink and downlink due to high annual rainfall rates for most of the stations. The overall result shows that the impact of heavy rainfall on satellite communication will be more severe in the Eastern part than the Western of Malaysia. [ABSTRACT FROM AUTHOR]

This paper describes the overview of Ku's spiritual literature on nursing through five stages in Taiwan: the development of a spiritual framework, a scale of spiritual distress, a spiritual care model, a spiritual nursing course, and a spiritual care book in nursing. The article demonstrates the process of building spiritual care in nursing from constructing theoretical frameworks, measurements, and applying to education. The integrative overview of Ku's spiritual studies developed in this article could be an example in clinical and education fields for nurse administrators to develop spiritual capabilities. [ABSTRACT FROM AUTHOR]

Background Several Kell-system antibodies are known to cause direct agglutination. Also, some specificities, such as anti-Ku, have been reported to react only via the indirect antiglobulin test (IAT). Methods Herein, we describe the case of a 61-year-old alloimmunized white woman who presented to an outside hospital with a gastrointestinal (GI) bleed and a "possible anti-Ku" was reported with 3+ reactivity at PEG-IAT and at Ficin-IAT; in addition to an unidentified cold antibody. Subsequently, when the patient presented to a second outside hospital, an anti-Ku that caused 3+ to 4+ reactions at saline-immediate spin (IS) was identified. The reactivity was evaluated with 0.01-M dithiothreitol (DTT) treatment of the plasma. Results It was determined that the strong agglutination with saline-IS was caused by immunoglobulin (Ig)M anti-Ku. Conclusion To our knowledge, this is the first reported case of an IgM anti-Ku. [ABSTRACT FROM AUTHOR]

DNA-binding proteins composed of DNA-binding domains directly affect genomic functions, mainly by performing transcription, DNA replication or DNA repair. Here, we briefly describe the DNA-binding proteins according to these three major functions. Transcription factors that usually bind to specific sequences of DNA could be classified based on their sequence similarity and the structure of the DNA-binding domains, such as basic, zinc-coordinating, helix-turn-helix domains, etc. Most DNA replication factors do not need a specific sequence of DNA, but instead mainly depend on a DNA structure, with the exception of the origin recognition complex in yeast or Escherichia coli that recognizes the DNA sequences at particular origins. DNA replication includes initiation and elongation. The major DNA-binding proteins involved in these two steps are briefly described. DNA repair proteins bound to DNA depend on the damaged DNA structure. They are classified to base excision repair, DNA mismatch repair, nucleotide excision repair, homologous recombination repair and non-homologous end joining. The major DNA-binding proteins involved in these pathways are briefly described. Histone and high mobility group are two examples of DNA-binding proteins that do not belong to the three categories above and are briefly described. Finally, we warn that the non-specific binding proteins might have an affinity to some non-specific medium materials such as protein A or G beads that are commonly used for immune precipitation, which can easily generate false positive signals while detecting protein-protein interaction; therefore, the results need to be carefully analyzed using positive/negative controls.

Radiotherapy is one of the mainstay treatments for hepatocellular carcinoma (HCC). However, a substantial number of patients with HCC develop radioresistance and eventually suffer from tumor progression or relapse, which is a major impediment to the use of radiotherapy. Therefore, elucidating the mechanisms underlying radioresistance and identifying novel therapeutic targets to improve patient prognosis are important in HCC management. In this study, using in vitro and in vivo models, laser microirradiation and live cell imaging methods, and coimmunoprecipitation assays, we report that a DNA repair enhancer, human positive cofactor 4 (PC4), promotes nonhomologous end joining-based DNA repair and renders HCC cells resistant to radiation. Mechanistically, PC4 interacts with poly (ADP-ribose) polymerase 1 and directs Ku complex PARylation, resulting in the successful recruitment of the Ku complex to damaged chromatin and increasing the efficiency of nonhomologous end joining repair. Clinically, PC4 is highly expressed in tumor tissues and is correlated with poor prognosis in patients with HCC. Taken together, our data suggest that PC4 is a DNA repair driver that can be targeted to radiosensitize HCC cells., Competing Interests: Conflict of interest All authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In Saccharomyces cerevisiae and in humans, the telomerase RNA subunit is bound by Ku, a ring-shaped protein heterodimer best known for its function in DNA repair. Ku binding to yeast telomerase RNA promotes telomere lengthening and telomerase recruitment to telomeres, but how this is achieved remains unknown. Using telomere-length analysis and chromatin immunoprecipitation, we show that Sir4 – a previously identified Ku-binding protein that is a component of telomeric silent chromatin – is required for Ku-mediated telomere lengthening and telomerase recruitment. We also find that specifically tethering Sir4 directly to Ku-binding-defective telomerase RNA restores otherwise-shortened telomeres to wild-type length. These findings suggest that Sir4 is the telomere-bound target of Ku-mediated telomerase recruitment and provide one mechanism for how the Sir4-competing Rif1 and Rif2 proteins negatively regulate telomere length in yeast.

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair processes, especially nonhomologous DNA-end joining (NHEJ), is critical for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, such as human Ku70 and Ku80, might play critical roles in controlling NHEJ activity. XRCC4-like factor (XLF) is a core NHEJ factor and plays a key role in the Ku-dependent NHEJ repair process in human cells. Recently, companion animals, such as canines, have been proposed to be a good model for many aspects of cancer research, including the development of chemotherapeutics. However, the localization and regulation of core NHEJ factors in canine cells have not been elucidated. Here, we show that the localization of canine XLF changes dynamically during the cell cycle. EYFP-canine XLF localizes in the nuclei of interphase cells and accumulates immediately at microirradiated DSB sites. The structure of a putative human XLF nuclear localization signal (NLS) and a putative 14-3-3 binding motif are evolutionarily conserved in canine, chimpanzee and mouse XLF. However, the putative β-TRCP-recognizable degron of human XLF is not conserved in canine and mouse. Additionally, some vital human XLF phosphorylation sites, including the ATM major phosphorylation site (S251), are not conserved in canine XLF. Our findings might be useful for the study of the molecular mechanisms of NHEJ in canine cells and for the development of new radiosensitizers that target XLF. [ABSTRACT FROM AUTHOR]

Various chemotherapies and radiation therapies are useful for killing cancer cells mainly by inducing DNA double-strand breaks (DSBs). Uncovering the molecular mechanisms of DSB repair processes is crucial for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. XRCC4 plays a critical role in Ku-dependent nonhomologous DNA-end joining (NHEJ) in human cells, and is one of the core NHEJ factors. The localization of core NHEJ factors, such as human Ku70 and Ku80, might play a crucial role in regulating NHEJ activity. Recently, companion animals, such as canines, have been proposed to be a good model in many aspects of cancer research. However, the localization and regulation mechanisms of core NHEJ factors in canine cells have not been elucidated. Here, we show that the expression and subcellular localization of canine XRCC4 changes dynamically during the cell cycle. Furthermore, EYFP-canine XRCC4 accumulates quickly at laser-microirradiated DSB sites. The structure of a putative human XRCC4 nuclear localization signal (NLS) is highly conserved in canine, chimpanzee and mouse XRCC4. However, the amino acid residue corresponding to the human XRCC4 K210, thought to be important for nuclear localization, is not conserved in canine XRCC4. Our findings might be useful for the study of the molecular mechanisms of Ku-dependent NHEJ in canine cells and the development of new radiosensitizers that target XRCC4. [ABSTRACT FROM AUTHOR]

Summary During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA. [ABSTRACT FROM AUTHOR]

Summary In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a c-NHEJ core component. We report here that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(D)J recombination assays, and do not shift toward higher micro-homology usage in plasmid repair assays. Although PAXX-deficient cells lack c-NHEJ phenotypes, PAXX forms a stable ternary complex with Ku bound to DNA. Formation of this complex involves an interaction with Ku70 and requires a bare DNA extension for stability. Moreover, the relatively weak Ku-dependent stimulation of LIG4/XRCC4 activity by PAXX is unmasked by XLF ablation. Thus, PAXX plays an accessory role during c-NHEJ that is largely overlapped by XLF’s function. [ABSTRACT FROM AUTHOR]

We aimed to study the prevalence and clinical associations of myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs) in a large cohort of Indian patients with idiopathic inflammatory myositis (IIM). Clinical details and serum samples were collected from patients with IIM (satisfying Bohan and Peter Criteria, 1975) and CTD-associated myositis. Sera were analysed for antibodies against SRP, Mi2, Jo1, PL7, PL12, EJ, OJ, Ro52, Ku, Pm-Scl 75 and PM-Scl 100, using immunoblot assay. The cohort comprised 124 patients with IIM (M:F = 1:3.6). Fifty-five of them had dermatomyositis (DM), 22 had juvenile dermatomyositis (JDM), 25 had polymyositis (PM) and 22 had connective tissue disease-associated myositis (CTD myositis). Mean disease duration was 10.9 months. ANA was positive in 84 (68.9 %), and MSAs in 61 (49.2 %) patients. Among MSAs, autoantibodies to Mi2, synthetase (Jo1, PL7, PL12, EJ) and SRP were present in 26 (20.9 %), 29 (23.4 %) and 6 (4.8 %) patients, respectively. Prevalence of MAAs was as follows: antibodies to Ro52 in 45 (36.3 %), Ku and PM-Scl 75 in 13 (10.5 %) and PM-Scl 100 in 5 (4 %) patients. Anti-Mi2 antibodies were positively associated with DM (21/55, 38.2 %; p < 0.0001) and pharyngeal weakness (13/34, 38.2 %; p = 0.004) and negatively associated with ILD (0/28; p = 0.001). ILD and mechanics' hands were significantly more in patients with anti-synthetase antibodies (16/28, 57 % and 14/22, 63.6 %; p < 0.0001). Four of six patients with anti-SRP antibody showed poor response to multiple drugs. Higher prevalence of anti-Mi2 is probably related to higher proportion of patients with DM. Absence of ILD in patients with anti-Mi2 antibody suggests that it may protect against ILD. In Indian population also, anti-synthetase antibodies are associated with ILD, and anti-SRP antibodies with poor response to treatment. [ABSTRACT FROM AUTHOR]

Background: Autoantibodies targeting Ku, an abundant nuclear protein with DNA helicase activity, have been reported in patients with systemic autoimmune rheumatic diseases. Little is known about the clinical associations of anti-Ku antibodies, especially when novel diagnostic technologies are used. The objective of the present study was to analyse the prevalence of anti- Ku antibodies in different medical conditions using a novel chemiluminescent immunoassay. Patients and methods: Serum samples from adult patients with systemic lupus erythematosus (SLE, n=305), systemic sclerosis (SSc, n=70) and autoimmune myositis patients (AIM, n=109) were the primary focus of the study. Results were compared with disease controls (rheumatoid arthritis, RA, n=30; infectious diseases, n=17) and healthy individuals (n=167). In addition, samples submitted for routine autoantibody testing from patients referred to a rheumatology clinic (n=1078) were studied. All samples were tested for anti-Ku antibodies by QUANTA Flash Ku chemiluminescent immunoassay (research use only, Inova Diagnostics, San Diego, USA) using full length recombinant human Ku. SLE patient samples were also tested for other autoantibodies. Clinical data of anti-Ku antibody positive patients (high titres) were obtained by retrospective chart review. Results and findings: In the disease cohorts, 30/305 (9.8%) SLE, 3/70 (4.3%) systemic sclerosis and 4/109 (3.7%) autoimmune myositis (AIM) patients were positive, respectively. The four positive AIM patients had an overlap myositis syndrome that included two patients with SLE. The three systemic sclerosis (SSc) positive samples had diagnoses of SSc/SLE overlap, diffuse cutaneous SSc, and early edematous phase SSc. In the control cohorts, 2/170 (1.2%) healthy individuals (all low titre), 0/30 (0.0%) (RA) and 0/17 (0.0%) infectious disease patients were positive. The area under the curve values were: 0.75 for SLE vs. controls, 0.68 for SSc vs. controls and 0.37 for AIM vs. controls. In the rheumatology clinic referral cohort, 12/1078 (1.1%) were positive for anti-Ku antibodies, nine showing low and three high titres. The diagnoses of the three high positive anti-Ku positive patients were: probable SLE, mixed connective tissue disease (MCTD) and ANA positive RA. Conclusion: Anti-Ku antibodies detected by chemiluminescent immunoassay are most prevalent in SLE. When found in AIM and SSc, they were associated with overlap syndrome and early SSc. [ABSTRACT FROM AUTHOR]

Ku heterodimer is a DNA binding protein with a prominent role in DNA repair. Here, we investigate whether and how Ku impacts the DNA damage response by acting as a post-transcriptional regulator of gene expression. We show that Ku represses p53 protein synthesis and p53-mediated apoptosis by binding to a bulged stem-loop structure within the p53 5′ UTR. However, Ku-mediated translational repression of the p53 mRNA is relieved after genotoxic stress. The underlying mechanism involves Ku acetylation which disrupts Ku-p53 mRNA interactions. These results suggest that Ku-mediated repression of p53 mRNA translation constitutes a novel mechanism linking DNA repair and mRNA translation. [ABSTRACT FROM AUTHOR]