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Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

Mammalian polynucleotide kinase 3'-phosphatase (PNKP) is a dual-function DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, which generate 3'-OH and 5'-phosphate termini respectively, as substrates for DNA polymerase and DNA ligase to complete DNA repair. PNKP is thus involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes, which involve distinct sets of proteins. In this study, we report that PNKP is acetylated at two lysine (K142 and K226) residues. While K142 (AcK142) is constitutively acetylated by p300, CBP acetylates K226 (AcK226) only after DSB induction. Co-immunoprecipitation analysis using antibodies specific for PNKP peptides containing AcK142 or AcK226 of PNKP showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP only with DSBR proteins. Although acetylation at these residues did not significantly affect the enzymatic activity of PNKP in vitro, cells expressing nonacetylable PNKP (K142R or K226R) accumulated DNA damage, specifically in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This observation is consistent with the reported degradation of CBP but not p300 in HD cells. Moreover, genomes of HD cells progressively accumulated DSBs specifically in the transcribed genes. Chromatin-immunoprecipitation analysis using anti-AcK142 or anti-AcK226 antibodies demonstrated an association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings collectively demonstrate that acetylation at two lysine residues located in different domains of PNKP regulates its functionally distinct role in BER/SSBR vs. DSBR.

Polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA end-processing enzyme in mammals with 3'-phosphatase and 5'-kinase activities, plays a pivotal role in multiple DNA repair pathways. Its functional deficiency has been etiologically linked to various neurological disorders. Recent reports have shown that mutation at a conserved glutamine (Gln) in PNKP leads to late-onset ataxia with oculomotor apraxia type 4 (AOA4) in humans and embryonic lethality in pigs. However, the molecular mechanism underlying such phenotypes remains elusive. Here, we report that the enzymatic activities of the mutant versus WT PNKP are comparable; however, cells expressing mutant PNKP and peripheral blood mononuclear cells (PBMCs) of AOA4 patients showed a significant amount of DNA double-strand break accumulation and consequent activation of the DNA damage response. Further investigation revealed that the nuclear localization of mutant PNKP is severely abrogated, and the mutant proteins remain primarily in the cytoplasm. Western blot analysis of AOA4 patient-derived PBMCs also revealed the presence of mutated PNKP predominantly in the cytoplasm. To understand the molecular determinants, we identified that mutation at a conserved Gln residue impedes the interaction of PNKP with importin alpha but not with importin beta, two highly conserved proteins that mediate the import of proteins from the cytoplasm into the nucleus. Collectively, our data suggest that the absence of PNKP in the nucleus leads to constant activation of the DNA damage response due to persistent accumulation of double-strand breaks in the mutant cells, triggering death of vulnerable brain cells-a potential cause of neurodegeneration in AOA4 patients.

A growing body of evidence indicates that RNA plays a critical role in orchestrating DNA double-strand break repair (DSBR). Recently, we showed that homologous nascent RNA can be used as a template for error-free repair of double-strand breaks (DSBs) in the transcribed genome and to restore the missing sequence at the break site via the transcription-coupled classical nonhomologous end-joining (TC-NHEJ) pathway. TC-NHEJ is a complex multistep process in which a reverse transcriptase (RT) is essential for synthesizing the DNA strand from template RNA. However, the identity of the RT involved in the TC-NHEJ pathway remained unknown. Here, we report that DNA polymerase eta (Pol η), known to possess RT activity, plays a critical role in TC-NHEJ. We found that Pol η forms a multiprotein complex with RNAP II and other TC-NHEJ factors, while also associating with nascent RNA. Moreover, purified Pol η, along with DSBR proteins PNKP, XRCC4, and Ligase IV can fully repair RNA templated 3'-phosphate-containing gapped DNA substrate. In addition, we demonstrate here that Pol η deficiency leads to accumulation of R-loops and persistent strand breaks in the transcribed genes. Finally, we determined that, in Pol η depleted but not in control cells, TC-NHEJ-mediated repair was severely abrogated when a reporter plasmid containing a DSB with several nucleotide deletion within the E. coli lacZ gene was introduced for repair in lacZ-expressing mammalian cells. Thus, our data strongly suggest that RT activity of Pol η is required in error-free DSBR.

SARS-CoV-2 infection-induced aggravation of host innate immune response not only causes tissue damage and multiorgan failure in COVID-19 patients but also induces host genome damage and activates DNA damage response pathways. To test whether the compromised DNA repair capacity of individuals modulates the severity of COVID-19 infection, we analyze DNA repair gene expression in publicly available patient datasets and observe a lower level of the DNA glycosylase NEIL2 in the lungs of severely infected COVID-19 patients. This observation of lower NEIL2 levels is further validated in infected patients, hamsters and ACE2 receptor-expressing human A549 (A549-ACE2) cells. Furthermore, delivery of recombinant NEIL2 in A549-ACE2 cells shows decreased expression of proinflammatory genes and viral E-gene, as well as lowers the yield of viral progeny compared to mock-treated cells. Mechanistically, NEIL2 cooperatively binds to the 5'-UTR of SARS-CoV-2 genomic RNA to block viral protein synthesis. Collectively, these data strongly suggest that the maintenance of basal NEIL2 levels is critical for the protective response of hosts to viral infection and disease.

Compromised DNA repair capacity of individuals could play a critical role in the severity of SARS-CoV-2 infection-induced COVID-19. We therefore analyzed the expression of DNA repair genes in publicly available transcriptomic datasets of COVID-19 patients and found that the level of NEIL2, an oxidized base specific mammalian DNA glycosylase, is particularly low in the lungs of COVID-19 patients displaying severe symptoms. Downregulation of pulmonary NEIL2 in CoV-2-permissive animals and postmortem COVID-19 patients validated these results. To investigate the potential roles of NEIL2 in CoV-2 pathogenesis, we infected Neil2-null (Neil2-/-) mice with a mouse-adapted CoV-2 strain and found that Neil2-/- mice suffered more severe viral infection concomitant with increased expression of proinflammatory genes, which resulted in an enhanced mortality rate of 80%, up from 20% for the age matched Neil2+/+ cohorts. We also found that infected animals accumulated a significant amount of damage in their lung DNA. Surprisingly, recombinant NEIL2 delivered into permissive A549-ACE2 cells significantly decreased viral replication. Toward better understanding the mechanistic basis of how NEIL2 plays such a protective role against CoV-2 infection, we determined that NEIL2 specifically binds to the 5'-UTR of SARS-CoV-2 genomic RNA and blocks protein synthesis. Together, our data suggest that NEIL2 plays a previously unidentified role in regulating CoV-2-induced pathogenesis, via inhibiting viral replication and preventing exacerbated proinflammatory responses, and also via its well-established role of repairing host genome damage.

Respiratory syncytial virus (RSV), along with other prominent respiratory RNA viruses such as influenza and SARS-CoV-2, significantly contributes to the global incidence of respiratory tract infections. These pathogens induce the production of reactive oxygen species (ROS), which play a crucial role in the onset and progression of respiratory diseases. However, the mechanisms by which viral RNA manages ROS-induced base oxidation remain poorly understood. Here, we reveal that 8-oxo-7,8-dihydroguanine (8-oxoGua) is not merely an incidental byproduct of ROS activity but serves as a strategic adaptation of RSV RNA to maintain genetic fidelity by hijacking the 8-oxoguanine DNA glycosylase 1 (OGG1). Through RNA immunoprecipitation and next-generation sequencing, we discovered that OGG1 binding sites are predominantly found in the RSV antigenome, especially within guanine-rich sequences. Further investigation revealed that viral ribonucleoprotein complexes specifically exploit OGG1. Importantly, inhibiting OGG1's ability to recognize 8-oxoGua significantly decreases RSV progeny production. Our results underscore the viral replication machinery's adaptation to oxidative challenges, suggesting that inhibiting OGG1's reading function could be a novel strategy for antiviral intervention. Author summary: Respiratory tract infections caused by RSV present a significant public health concern, especially affecting infants and the elderly. Effective antiviral strategies are urgently needed against this non-segmented, negative-sense RNA virus of the Pneumoviridae family. Our research reveals how RSV maintains replication fitness despite oxidative damage, a common exacerbator of respiratory conditions. We have identified a mechanism where RSV preserves its genome integrity under oxidative stress using the DNA repair enzyme OGG1. This enzyme ensures that the viral RNA accurately pairs 8-oxoGua with cytosine, crucial for maintaining the fidelity of the viral genome. Targeting the interplay between OGG1 and RSV RNA offers a promising approach for developing novel antiviral treatments, potentially countering the virus's evolutionary adaptations. [ABSTRACT FROM AUTHOR]

As 5G mobile systems carry multiple services and applications, numerous user, and application types with varying quality of service requirements inside a single physical network infrastructure are the primary problem in constructing 5G networks. Radio Access Network (RAN) slicing is introduced as a way to solve these challenges. This research focuses on optimizing RAN slices within a singular physical cell for vehicle-to-everything (V2X) and enhanced mobile broadband (eMBB) UEs, highlighting the importance of adept resource management and allocation for the evolving landscape of 5G services. We put forth two unique strategies: one being offline network slicing, also referred to as standard network slicing, and the other being Online reinforcement learning (RL) network slicing. Both strategies aim to maximize network efficiency by gathering network model characteristics and augmenting radio resources for eMBB and V2X UEs. When compared to traditional network slicing, RL network slicing shows greater performance in the allocation and utilization of UE resources. These steps are taken to adapt to fluctuating traffic loads using RL strategies, with the ultimate objective of bolstering the efficiency of generic 5G services. [ABSTRACT FROM AUTHOR]

The accelerated growth of 5G technology has facilitated substantial progress in the realm of vehicle-to-everything (V2X) communications. Consequently, achieving optimal network performance and addressing congestion-related challenges have become paramount. This research proposes a unique hybrid power and rate control management strategy for distributed congestion control (HPR-DCC) focusing on 5G-NR-V2X sidelink communications. The primary objective of this strategy is to enhance network performance while simultaneously preventing congestion. By implementing the HPR-DCC strategy, a more fine-grained and adaptive control over the transmit power and transmission rate can be achieved. This enables efficient control by dynamically adjusting transmission parameters based on the network conditions. This study outlines the system model and methodology used to develop the HPR-DCC algorithm and investigates its characteristics of stability and convergence. Simulation results indicate that the proposed method effectively controls the maximum CBR value at 64% during high congestion scenarios, which leads to a 6% performance improvement over the conventional DCC approach. Furthermore, this approach enhances the signal reception range by 20 m, while maintaining the 90% packet reception ratio (PRR). The proposed HPR-DCC contributes to optimizing the quality and reliability of 5G-NR-V2X sidelink communication and holds great promise for advancing V2X applications in intelligent transportation systems. [ABSTRACT FROM AUTHOR]

Importance: Bangladesh hosts a large number of Rohingya refugees from Myanmar. Living in refugee camps, the Rohingya refugees face challenges in everyday occupations because of violence, limited opportunities, and corporal punishment by the community. Objective: To explore how Rohingya refugees experience participation in everyday occupations while living in temporary refugee camps in Bangladesh. Design: Phenomenological study to describe, understand, and interpret the meanings of life experiences in particularly adverse conditions. Setting: Rohingya refugee camps in Bangladesh. Participants: Fifteen purposively selected participants from the camps. Outcomes and Measures: In-depth semistructured interview, as well as participant and environmental observations. Researchers used line-by-line data analysis to capture quotations and patterns using interpretive phenomenological analysis, which included establishment of initial codes, interpretation, determining selected codes, and categorization. Results: The research identified four major themes—(1) mental stress, sleep disturbances, and daily occupations; (2) adjustment to inconsistent daily activities; (3) complex relationships and limited social roles that decreased occupational engagement; and (4) involvement in precarious occupations that exacerbated severe health risks—and four subthemes—(1) fragmented family relationships, (2) formation of new relationships to perform social roles, (3) inconvenient and inaccessible living conditions, and (4) continuation of unlawful work to survive. Conclusions and Relevance: Rohingya refugees should receive comprehensive health and rehabilitative care because of their perilous mental health conditions, precarious occupations, and lack of trustworthy relationships with family and neighbors. What This Article Adds: Rohingya refugees experience imbalanced, deprived, and maladapted occupations in refugee camps. Suggestions to improve their lived experience with further peer support programs may help them participate in occupation-based rehabilitation services to facilitate their social integration. Rohingya refugees experience imbalanced, deprived, and maladapted occupations in refugee camps and should have access to comprehensive health and rehabilitative care, including occupation-based services, to facilitate their social integration. [ABSTRACT FROM AUTHOR]

The recently explained cytokine, which is produced after the stimulation of interferon (IFN)-c, interleukin (IL)-2, and IL-18 is IL-32, has pro)inflammatory IFN-c, IL-2 and IL-18 are IL-32 mediator’s properties that are generally entailed in many diseases, including infections, cancer, and chronic inflammation. After the initial statement in 2005, it promoted the osteoclast precursor’s differentiation into TRAcP plus VNR plus multinucleated cells that express explicit osteoclast indicators. Furthermore, the loss of bone resorption might be accredited because of the collapse of the multinucleated cells, which are produced of the reaction to IL-32 to direct factoring that is ultimately essential for attaching the cells for bone resorption. Thus, in conclusion, IL-32, the pro-inflammatory mediator, has an important and indirect role in regulating osteoclast differentiation. In bone disorder’s pathophysiology, critical role of IL-32 needs more scientific evidence to develop a rational treatment protocol. IL-32 can become a potent mediator of active osteoclast generation in the presence of receptor activator of NF-κB ligand (RANKL). This novel cytokine can introduce more favorable conditions for osteoclastogenesis in the rheumatic arthritis by increasing the RANKL and osteoprotegerin ratio in fibroblast-like synoviocytes. [ABSTRACT FROM AUTHOR]