Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps.

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

How Does Literacy Affect Speech Processing? Not by Enhancing Cortical Responses to Speech, But by Promoting Connectivity of Acoustic-Phonetic and Graphomotor Cortices.

Previous research suggests that literacy, specifically learning alphabetic letter-to-phoneme mappings, modifies online speech processing and enhances brain responses, as indexed by the BOLD, to speech in auditory areas associated with phonological processing (Dehaene et al., 2010). However, alphabets are not the only orthographic systems in use in the world, and hundreds of millions of individuals speak languages that are not written using alphabets. In order to make claims that literacy per se has broad and general consequences for brain responses to speech, one must seek confirmatory evidence from nonalphabetic literacy. To this end, we conducted a longitudinal fMRI study in India probing the effect of literacy in Devanagari, an abubgida, on functional connectivity and cerebral responses to speech in 91 variously literate Hindi-speaking male and female human participants. Twenty-two completely illiterate participants underwent 6 months of reading and writing training. Devanagari literacy increases functional connectivity between acoustic-phonetic and graphomotor brain areas, but we find no evidence that literacy changes brain responses to speech, either in cross-sectional or longitudinal analyses. These findings shows that a dramatic reconfiguration of the neurofunctional substrates of online speech processing may not be a universal result of learning to read, and suggest that the influence of writing on speech processing should also be investigated.SIGNIFICANCE STATEMENT It is widely claimed that a consequence of being able to read is enhanced auditory processing of speech, reflected by increased cortical responses in areas associated with phonological processing. Here we find no relationship between literacy and the magnitude of brain response to speech stimuli in individuals who speak Hindi, which is written using a nonalphabetic script, Devanagari, an abugida. We propose that the exact nature of the script under examination must be considered before making sweeping claims about the consequences of literacy for the brain. Further, we find evidence that literacy enhances functional connectivity between auditory processing areas and graphomotor areas, suggesting a mechanism whereby learning to write might influence speech perception.

Abrogation of FBW7α-dependent p53 degradation enhances p53's function as a tumor suppressor.

The gene encoding the tumor suppressor p53 is mutated in most cancers. p53 expression is known to be tightly controlled by several E3 ligases. Here, we show that F-box and WD repeat domain-containing 7α (FBW7α), the substrate-recognition component of the SCFFBW7 multiprotein E3 ligase complex, targets both WT and tumor-derived mutants of p53 for proteasomal degradation in multiple human cancer cell lines (HCT116 and U2OS). We found that lack of FBW7α stabilizes p53 levels, thereby increasing its half-life. p53 ubiquitylation and subsequent degradation require the F-box and the C-terminal WD40 repeats in FBW7α. The polyubiquitylation of p53 occurred via Lys-48 linkage and involved phosphorylation on p53 at Ser-33 and Ser-37 by glycogen synthase kinase 3ß (GSK3ß) and DNA-dependent protein kinase (DNA-PK), respectively. These phosphorylation events created a phosphodegron that enhanced p53 binding to FBW7α, allowing for the attachment of polyubiquitin moieties at Lys-132 in p53. FBW7α-dependent p53 polyubiquitylation apparently occurred during and immediately after DNA double-strand breaks induced by either doxorubicin or ionizing radiation. Accordingly, in cells lacking FBW7α, p53 induction was enhanced after DNA damage. Phosphodegron-mediated polyubiquitylation of p53 on Lys-132 had functional consequences, with cells in which FBW7α-mediated p53 degradation was abrogated exhibiting enhancement of their tumorigenic potential. We conclude that p53, which previously has been reported to transactivate FBW7, is also targeted by the same E3 ligase for degradation, suggesting the presence of a regulatory feedback loop that controls p53 levels and functions during DNA damage.

Assessment of Lipid Peroxidation Levels and Total Antioxidant Status in Chronic and Aggressive Periodontitis Patients: An in vivo Study.

INTRODUCTION: Periodontitis is a common problem affecting a significant population of the world. For the assessment of oxidative stress of an individual, total oxidation status (TOS) and total antioxidant capacity (TAOC) are the significant biomarkers. Hence, we planned the present study to assess malondialde-hyde (MDA), TOS, TAOC levels, and oxidative stress index (OSI) in generalized aggressive periodontitis (GP) and chronic periodontitis (CP) patients. MATERIALS AND METHODS: The present study included assessment of 40 CP patients, 40 GP patients, and 40 healthy controls. Clinical assessment of all the subjects was done by measuring the probing depth (PD), clinical attachment (CL), gingival index (GI), gingival bleeding index (GBI), and plaque index (PI). Salivary and serum samples were taken and assessed by standard procedures as described previously in the literature. All the values were assessed and compared. RESULTS: Significant results were obtained while comparing all the periodontal parameters in between various study groups. Mean serum MDA levels in the CP, GP, and control group were found to be 0.68, 0.65, and 0.61 µM respectively. Statistically nonsignificant results were obtained while comparing the serum MDA levels in between the three study groups. Significant results were obtained while comparing the mean serum and salivary TOS values, TAOC values, and OSI in between various study groups. CONCLUSION: In periodontitis patients, oxidative stress was significantly higher in comparison with healthy subjects. CLINICAL SIGNIFICANCE: Oxidative parameters do play a significant role in the pathologic profile of periodontitis.

BLM helicase-dependent and -independent roles of 53BP1 during replication stress-mediated homologous recombination.

Mutations in BLM helicase cause Bloom syndrome, characterized by predisposition to all forms of cancer. We demonstrate that BLM, signal transducer 53BP1, and RAD51 interact during stalled replication. Interactions between the three proteins have functional consequences. Lack of 53BP1 decreases the cell survival and enhanced chromosomal aberration after replication arrest. 53BP1 exhibits both BLM-dependent and -independent anti-recombinogenic functions in human and mouse cells. Both BLM and 53BP1 abrogate endogenous RAD51 foci formation and disrupt RAD51 polymerization. Consequently, loss of BLM and 53BP1 synergistically enhances stress-dependent homologous recombination. These results provide evidence regarding the cooperation between BLM and 53BP1 during maintenance of genomic integrity.

BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54.

Mutation of BLM helicase results in the autosomal recessive disorder Bloom syndrome (BS). Patients with BS exhibit hyper-recombination and are prone to almost all forms of cancer. BLM can exhibit its anti-recombinogenic function either by dissolution of double Holliday junctions or by disruption of RAD51 nucleofilaments. We have now found that BLM can interact with the pro-recombinogenic protein RAD54 through an internal ten-residue polypeptide stretch in the N-terminal region of the helicase. The N-terminal region of BLM prevented the formation of RAD51-RAD54 complex, both in vitro and in vivo. Using the fluorescence recovery after photobleaching (FRAP) technique, we found that RAD54 and BLM rapidly and concurrently, yet transiently, bound to the chromatinized foci. Presence of BLM enhanced the mobility of both soluble and chromatinized RAD51 but not RAD54. The BLM-RAD54 interaction could occur even in absence of functional RAD51. The N-terminal 1-212 amino acids of BLM or an ATPase-dead mutant of the full-length helicase enhanced the ATPase and chromatin-remodeling activities of RAD54. These results indicate that apart from its dominant function as an anti-recombinogenic protein, BLM also has a transient pro-recombinogenic function by enhancing the activity of RAD54.

RIF1 promotes replication fork protection and efficient restart to maintain genome stability.

Homologous recombination (HR) and Fanconi Anemia (FA) pathway proteins in addition to their DNA repair functions, limit nuclease-mediated processing of stalled replication forks. However, the mechanism by which replication fork degradation results in genome instability is poorly understood. Here, we identify RIF1, a non-homologous end joining (NHEJ) factor, to be enriched at stalled replication forks. Rif1 knockout cells are proficient for recombination, but displayed degradation of reversed forks, which depends on DNA2 nuclease activity. Notably, RIF1-mediated protection of replication forks is independent of its function in NHEJ, but depends on its interaction with Protein Phosphatase 1. RIF1 deficiency delays fork restart and results in exposure of under-replicated DNA, which is the precursor of subsequent genomic instability. Our data implicate RIF1 to be an essential factor for replication fork protection, and uncover the mechanisms by which unprotected DNA replication forks can lead to genome instability in recombination-proficient conditions.

Phosphorylation-dependent interactions of BLM and 53BP1 are required for their anti-recombinogenic roles during homologous recombination.

Mutations in bloom helicase protein (BLM) helicase cause Bloom syndrome, characterized by predisposition to almost all forms of cancer. We have demonstrated previously that endogenous BLM, signal transducer 53BP1 and RAD51 are present in a complex during replication stress. Using full-length recombinant proteins, we now provide evidence that these proteins physically interact. BLM interacts with checkpoint kinase (Chk) 1 via the kinetochore-binding domain (KBD). Wild-type (WT) Chk1 phosphorylates 53BP1 in the KBD, both in vitro and in vivo during replication stress. Chk1-mediated phosphorylation of 53BP1 enhances its binding to BLM and is required for the accumulation of 53BP1 at the site of stalled replication. 53BP1, in turn, binds to the N-terminal domain of BLM. Ataxia telangiectasia and Rad3 related (ATR)-mediated phosphorylation of BLM at Thr99 is critical for its interaction and subsequent co-localization with 53BP1. WT BLM enhances the interaction and co-localization between 53BP1 and RAD51 during replication arrest. Interactions between the three proteins have functional consequences. Non-binding or phosphorylation-deficient mutants of BLM and 53BP1 fail to demonstrate the anti-recombinogenic property of the WT counterparts. Consequently, these mutants cause elevation of endogenous RAD51 foci formation. These results provide evidence that the phosphorylation-mediated interactions between BLM, 53BP1 and RAD51 are required for their regulatory roles during homologous recombination.

MRN complex-dependent recruitment of ubiquitylated BLM helicase to DSBs negatively regulates DNA repair pathways.

Mutations in BLM in Bloom Syndrome patients predispose them to multiple types of cancers. Here we report that BLM is recruited in a biphasic manner to annotated DSBs. BLM recruitment is dependent on the presence of NBS1, MRE11 and ATM. While ATM activity is essential for BLM recruitment in early phase, it is dispensable in late phase when MRE11 exonuclease activity and RNF8-mediated ubiquitylation of BLM are the key determinants. Interaction between polyubiquitylated BLM and NBS1 is essential for the helicase to be retained at the DSBs. The helicase activity of BLM is required for the recruitment of HR and c-NHEJ factors onto the chromatin in S- and G1-phase, respectively. During the repair phase, BLM inhibits HR in S-phase and c-NHEJ in G1-phase. Consequently, inhibition of helicase activity of BLM enhances the rate of DNA alterations. Thus BLM utilizes its pro- and anti-repair functions to maintain genome stability.

BLM Potentiates c-Jun Degradation and Alters Its Function as an Oncogenic Transcription Factor.

Mutations in BLM helicase predispose Bloom syndrome (BS) patients to a wide spectrum of cancers. We demonstrate that MIB1-ubiquitylated BLM in G1 phase functions as an adaptor protein by enhancing the binding of transcription factor c-Jun and its E3 ligase, Fbw7α. BLM enhances the K48/K63-linked ubiquitylation on c-Jun, thereby enhancing the rate of its subsequent degradation. Functionally defective Fbw7α mutants prevalent in multiple human cancers are reactivated by BLM. However, BS patient-derived BLM mutants cannot potentiate Fbw7α-dependent c-Jun degradation. The decrease in the levels of c-Jun in cells expressing BLM prevents effective c-Jun binding to 2,584 gene promoters. This causes decreases in the transcript and protein levels of c-Jun targets in BLM-expressing cells, resulting in attenuated c-Jun-dependent effects during neoplastic transformation. Thus, BLM carries out its function as a tumor suppressor by enhancing c-Jun turnover and thereby preventing its activity as a proto-oncogene.

A cross-sectional study on oral health status of battery factory workers in Chennai city.

AIM AND OBJECTIVE: Some occupational exposures are associated with oral changes in both hard and soft tissues. Presence of oral lesions can interfere with speech, swallowing, and general health of a patient. The present cross-sectional study was conducted to evaluate the oral health status of battery factory workers in Chennai city. MATERIALS AND METHODS: A total of 600 subjects were selected in battery factory out of 3500 workers using statistical sample selection formula 4pq/l(2) and divided into study and control groups based on acid exposure. The data were recorded on a modified World Health Organization 1997 pro forma. The data were evaluated using Chi-square test and Mann-Whitney U-test. MATERIALS AND METHODS: A total of 600 subjects were selected in battery factory out of 3500 workers using statistical sample selection formula 4pq/l(2) and divided into study and control groups based on acid exposure. The data were recorded on a modified World Health Organization 1997 pro forma. The data were evaluated using Chi-square test and Mann-Whitney U-test. RESULTS: Oral symptoms such as disturbed taste, dry mouth, oral ulcers, and foul breath were statistically significant between the groups (<0.001). Dental erosion was statistically significant with the duration of working years. Dental erosion was significant among study group compared to control (0.001). CONCLUSION: The present study showed that selected samples had various oral conditions due to exposure to acids from battery. It was observed that oral health problems were directly related to the duration of acid exposure in the study group. Implementing exhaust ventilation and monitoring the devices help in reducing the acid exposure. Implementation of oral hygiene education and nutritional supplementation helps in improving their oral health.

Evaluation of the efficacy of platelet-rich plasma and platelet-rich fibrin in alveolar defects after removal of impacted bilateral mandibular third molars.

AIM AND OBJECTIVES: This study attempted the evaluation of the efficacy of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in alveolar defects after removal of bilateral mandibular third molars. MATERIALS AND METHODS: A total of 30 patients reporting to Department of Oral and Maxillofacial Surgery and having bilateral mandibular third molar impaction in both male and female aged between 18 and 30 years were included in this study. PRF and PRP were placed in extraction site and recalled at 2(nd), 4(th), and 6(th) month postoperatively. Data were statistically analyzed using IBM SPSS software for Windows, version 19.0. IBM Corp., Armonk, NY, USA. RESULTS: This study showed decreased probing depth in PRF group compared to PRP and control one. This signifies a better soft tissue healing of extraction sockets with PRF as compared to the PRP and the control group and increase in the bone density highlights the use of PRP and PRF certainly as a valid method in inducing hard tissue regeneration. CONCLUSION: This study indicates a definite improvement in the periodontal health distal to second molar after third molar surgery in cases treated with PRF as compared to the PRP group and control group. Hence, PRP and PRF can be incorporated as an adjunct to promote wound healing and osseous regeneration in mandibular third molar extraction sites.