The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.

Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.

Feedback-regulated transcriptional repression of FBXO31 by c-Myc triggers ovarian cancer tumorigenesis.

FBXO31, a member of F-box protein family, has been shown to play an important role in preventing tumorigenesis by preserving genomic stability during cell proliferation as well as upon genotoxic stress. Inactivation of FBXO31 due to loss of heterozygosity is associated with various cancers, including ovarian cancer, one of the deadliest forms of gynecological cancers. However, the role and regulation of FBXO31 in ovarian cancer remained elusive. Here, using biochemical and molecular biology techniques, we show that c-Myc suppresses the mRNA levels of FBXO31 in ovarian cancer. Chromatin immunoprecipitation experiment showed that c-Myc is recruited to the promoter region of FBXO31 and prevents FBXO31 mRNA synthesis. In contrast, FBXO31 maintains the c-Myc expression at an optimum through proteasome pathway. FBXO31 interacts with and facilitates the polyubiquitination of c-Myc through the SCF complex and thereby inhibits ovarian cancer growth both in vitro and in vivo. Moreover, FBXO31-mediated proteasomal degradation of c-Myc is unique. Unlike other negative regulators, FBXO31 recognizes c-Myc in phosphorylation independent manner to direct its degradation. Further, expression levels analysis revealed that c-Myc and FBXO31 share a converse correlation of expression in ovarian cancer cell lines and patient samples. We observed an increase in the expression levels of c-Myc with a concomitant decrease in the levels of FBXO31 in higher grades of ovarian cancer patient samples. In conclusion, our study demonstrated that oncogene c-Myc impairs the tumor-suppressive functions of FBXO31 to promote ovarian cancer progression, and therefore c-Myc-FBXO31 axis can be explored to develop better cancer therapy.

Co-operative binding of SKP1, Cullin1 and Cullin7 to FBXW8 results in Cullin1-SKP1-FBXW8-Cullin7 functional complex formation that monitors cellular function of ß-TrCP1.

F-box protein FBXW8 is known to interact with scaffolding protein Cullin1 and Cullin7 to form SCF (SKP1, Cullin and F-box protein) complex. However, detail understanding about the importance of both Cullins for SCF-FBXW8 complex formation as well as its ubiquitin ligase activity remains elusive. Here, we show that, through in vitro and in vivo studies, Cullin1 and Cullin7 increase each other's binding to FBXW8 synergistically. Interestingly, absence of either Cullin results in abrogation of binding of other Cullin to FBXW8. Binding of SKP1 to FBXW8 also increases in the presence of both the Cullins. Thus, SKP1, Cullin1 and Cullin7 are essential to form Cullin1-SKP1-FBXW8-Cullin7 functional ubiquitin ligase complex. Further, using computational, mutational and biochemical analysis, we found that Cullin1 binds to N-terminus of FBXW8 through SKP1 while Cullin7 associates with C-terminus of FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex in a cooperative manner. Results showed that Cullin1-SKP1-FBXW8-Cullin7 complex plays a key role in maintaining the basal level expression of ß-TrCP1. Moreover, Cullin1-SKP1-FBXW8-Cullin7 complex promotes cell migration by activating ß-catenin via directing proteasomal degradation of ß-TrCP1. Overall, our study reveals the intriguing molecular mechanism of assembly of SKP1, Cullin1, Cullin7 and FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex that control the function of ß-TrCP1.

Back to the basics: Study of portable chest radiographic findings in 116 COVID-19 positive patients in an Indian tertiary care hospital.

CONTEXT: Paucity of literature of portable CXR findings in COVID-19. AIMS: Evaluate radiographic findings in COVID-19 patients and calculate sensitivity of radiographs with RT-PCR as gold standard. SUBJECTS AND METHODS: Total 116 COVID-19 patients underwent portable CXR between April-June, 2020. Two radiologists reviewed radiographs with respect to laterality, craniocaudal, mediolateral distribution, shape, density, unifocality/multifocality and number of lung zones. Sensitivity of radiography was calculated with RT-PCR as gold standard. STATISTICAL ANALYSIS USED: IBM SPSS Statistics Subscription software (IBM, New York, USA). RESULTS: Many patients 67.2% (78/116) were asymptomatic. Cough (21.5%, 25/116) and fever (17.6%, 20/116) were the most frequent symptoms. 36.2% (42/116) patients revealed COVID-19 pneumonia-like abnormalities on CXR. Sensitivity of CXR with RT-PCR as gold standard was 36.2% (CI: Confidence interval = 27.46% - 44.95%). More patients in symptomatic group (68.4%, 26/38) had abnormal CXR compared to asymptomatic group (20.5%, 16/78) [P < 0.0001]. Radiographs revealed both unilateral (57.1%, 24/42), bilateral (42.8%, 18/42), GGO (80.9%, 34/42), or consolidation (11/42, 26.1%) in a middle (57.1%, 24/42), lower zone (83.3%, 35/42) and peripheral distribution (78.5%, 33/42). Lesions were commonly patchy (88%, 37/42) and multifocal (59.5%, 25/42). Majority had single (40.4%, 17/42) or two zone (35.7%, 15/42) involvement. CONCLUSIONS: Significant number of COVID-19 patients were asymptomatic. Over 1/3rd of patients showed radiographic abnormalities. Symptomatic patients were more likely to show radiographic findings than asymptomatic patients. If radiographs identify pneumonia in appropriate clinical setting, CT can be avoided. Common radiographic abnormalities among COVID 19 patients were bilateral/unilateral, patchy, multifocal, ground glass opacity or consolidation in peripheral and middle/lower zone distribution.

ß-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of ß-TrCP2 in response to genotoxic stress.

F-box proteins ß-TrCP1 and ß-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of ß-TrCP1 and ß-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remain largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that ß-TrCP1 and ß-TrCP2 communicate during genotoxic stress. We found that expression levels of ß-TrCP1 are significantly increased while levels of ß-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of ß-TrCP1 and ß-TrCP2 via the phosphorylation of ß-TrCP1 at Ser158. Phosphorylated ß-TrCP1 potently promotes the proteasomal degradation of ß-TrCP2 and MDM2, resulting in the activation of p53. Additionally, ß-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by ß-TrCP2. Thus, ß-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of ß-TrCP2. Collectively, our findings elucidate an intriguing posttranslational regulatory mechanism of these two paralogs under genotoxic stress and revealed ß-TrCP1 as a key player in maintaining the genome integrity through the attenuation of ß-TrCP2 levels in response to genotoxic stress.

FBXW8 regulates G1 and S phases of cell cycle progression by restricting ß-TrCP1 function.

Sequential alteration in the expression levels of cell cycle regulatory proteins is crucial for faithful cell cycle progression to maintain the cellular homeostasis. F-box protein ß-TrCP1 is known to control the expression levels of several important cell cycle regulatory proteins. However, how the function of ß-TrCP1 is regulated in spatiotemporal manner during cell cycle progression remains elusive. Here, we show that expression levels of ß-TrCP1 oscillate during cell cycle progression with a minimum level at the G1 and S phases of cell cycle. Using biochemical, flow cytometry, and immunofluorescence techniques, we found that oscillation of ß-TrCP1 expression is controlled by another F-box protein FBXW8. FBXW8 directs the proteasomal degradation of ß-TrCP1 in MAPK pathway-dependent manner. Interestingly, we found that the attenuation of ß-TrCP1 by FBXW8 is important for Cdc25A-mediated cell cycle transition from G1 phase to S phase as well as DNA damage-free progression of S phase. Overall, our study reveals the intriguing molecular mechanism and significance of maintenance of ß-TrCP1 levels during cell cycle progression by FBXW8-mediated proteasomal degradation.

Effect of dexmedetomidine on intraocular pressure as an additive in peribulbar block during glaucoma surgery.

Purpose: The purpose of this study is to assess the effect of dexmedetomidine on intraocular pressure (IOP) as an additive in peribulbar injections in glaucoma surgeries. Methods: A prospective, randomized, double-blind, parallel assignment interventional study was conducted for patients undergoing glaucoma surgeries at a tertiary eye care hospital in North India. Patients were randomized to two groups, Dexmed group and Placebo group. In the Dexmed group, dexmedetomidine (0.4 µg/kg body weight) was given as an additive along with peribulbar block. The primary outcome was change in IOP pre- and postperibulbar injections (IOP before the block, and after 5 and 15 min of the block). Secondary outcome measures were onset of block, adverse effects (bradycardia, hypotension, respiratory depression, and level 4 sedation), and surgeon satisfaction. Results: A total of 104 patients were randomized, 52 each in the Dexmed group and Placebo group. The percentage decrease in IOP was significantly more in the Dexmed group than in the Placebo group both at 5 and 15 min' post block (P < 0.05). At 5 min, the mean percent decrease in IOP in Dexmed group was -10.48, whereas it was 2.85 in the Placebo group. At 15 min, the mean percent decrease in IOP was -22.59 and -9.42 in the Dexmed and Placebo group, respectively. There was no significant difference between the two groups in the onset of block and adverse effects. Surgeon satisfaction was significantly greater in the Dexmed group than the Placebo group (P < 0.05). Conclusion: Dexmedetomidine lowers IOP significantly in patients undergoing glaucoma surgeries with safe hemodynamic changes and sedative effect.

Generic and vision related quality of life associated with different types of cataract surgeries and different types of intraocular lens implantation.

OBJECTIVES: To assess the effects of different types of cataract surgeries and intraocular lenses on generic as well as vision related quality of life of cataract patients, using EQ-5D and IND-VFQ 33 instruments respectively. METHODS: An observational, longitudinal study of patients undergoing cataract surgery was carried out at three ophthalmology centres. Patients were prospectively admitted for surgery for age-related cataract. Generic quality of life was assessed by using Euroqol's EQ5D-5L questionnaire and vision related quality of life was assessed by the IND-VFQ-33 questionnaire. Data pertaining to vision function and quality of life were collected pre surgery and 4 weeks after the surgery. RESULTS: Out of total patients (n = 814) recruited for the study, 517 patients were interviewed for both pre-surgery and post-surgery for EQ5D and 519 patients were interviewed for both pre-surgery and post-surgery for IND VFQ 33 tool. The combined data from all three centres showed that Quality Adjusted Life Year (QALY) gains observed in patients undergoing phacoemulsification with foldable lens implantation (2.25 QALY) were significantly higher (0.57 QALY) as compared to Small Incision Cataract Surgery (SICS) with PMMA lens implantation (1.68 QALY). Highest improvement however, in all three subscales of IND-VFQ-33 tool were clearly observed for SICS with PMMA lens implantation. CONCLUSIONS: The study has elicited the Health related and vision related Quality of Life scores for cataract surgeries and subsequent lens implantation. This study also offers Health State Utility Values along with visual outcomes for different surgical procedures, lenses and for the combination of surgery with lens implantation for cataract procedures providing a useful resource for future economic evaluation studies.

Successful endovascular treatment of spontaneous intrarenal pseudoaneurysm in a case of tuberous sclerosis.

Tuberous sclerosis is a complex disorder which has multisystem involvement and varied clinical manifestations. Almost half of the patients have associated angiomyolipoma which contains fat, vascular and smooth muscle components. Spontaneous pseudoaneurysm formation is a complication of angiomyolipoma. Here we present a case of a female child presenting with hematuria who after thorough clinical and radiological investigations was diagnosed as a case of tuberous sclerosis with right intrarenal pseudoaneurysm. She was successfully treated with endovascular coil embolization.

The tumor suppressor FBXO31 preserves genomic integrity by regulating DNA replication and segregation through precise control of cyclin A levels.

F-box protein 31 (FBXO31) is a reported putative tumor suppressor, and its inactivation due to loss of heterozygosity is associated with cancers of different origins. An emerging body of literature has documented FBXO31's role in preserving genome integrity following DNA damage and in the cell cycle. However, knowledge regarding the role of FBXO31 during normal cell-cycle progression is restricted to its functions during the G2/M phase. Interestingly, FBXO31 levels remain high even during the early G1 phase, a crucial stage for preparing the cells for DNA replication. Therefore, we sought to investigate the functions of FBXO31 during the G1 phase of the cell cycle. Here, using flow cytometric, biochemical, and immunofluorescence techniques, we show that FBXO31 is essential for maintaining optimum expression of the cell-cycle protein cyclin A for efficient cell-cycle progression. Stable FBXO31 knockdown led to atypical accumulation of cyclin A during the G1 phase, driving premature DNA replication and compromised loading of the minichromosome maintenance complex, resulting in replication from fewer origins and DNA double-strand breaks. Because of these inherent defects in replication, FBXO31-knockdown cells were hypersensitive to replication stress-inducing agents and displayed pronounced genomic instability. Upon entering mitosis, the cells defective in DNA replication exhibited a delay in the prometaphase-to-metaphase transition and anaphase defects such as lagging and bridging chromosomes. In conclusion, our findings establish that FBXO31 plays a pivotal role in preserving genomic integrity by maintaining low cyclin A levels during the G1 phase for faithful genome duplication and segregation.

The SCFFBXO46 ubiquitin ligase complex mediates degradation of the tumor suppressor FBXO31 and thereby prevents premature cellular senescence.

The tumor suppressor F-box protein 31 (FBXO31) is indispensable for maintaining genomic stability. Its levels drastically increase following DNA damage, leading to cyclin D1 and MDM2 degradation and G1 and G2/M arrest. Prolonged arrest in these phases leads to cellular senescence. Accordingly, FBXO31 needs to be kept at low basal levels in unstressed conditions for normal cell cycle progression during growth and development. However, the molecular mechanism maintaining these basal FBXO31 levels has remained unclear. Here, we identified the F-box family SCF-E3 ubiquitin ligase FBXO46 (SCFFBXO46) as an important proteasomal regulator of FBXO31 and found that FBXO46 helps maintain basal FBXO31 levels under unstressed conditions and thereby prevents premature senescence. Using molecular docking and mutational studies, we showed that FBXO46 recognizes an RXXR motif located at the FBXO31 C terminus to direct its polyubiquitination and thereby proteasomal degradation. Furthermore, FBXO46 depletion enhanced the basal levels of FBXO31, resulting in senescence induction. In response to genotoxic stress, ATM (ataxia telangiectasia-mutated) Ser/Thr kinase-mediated phosphorylation of FBXO31 at Ser-278 maintained FBXO31 levels. In contrast, activated ATM phosphorylated FBXO46 at Ser-21/Ser-67, leading to its degradation via FBXO31. Thus, ATM-catalyzed phosphorylation after DNA damage governs FBXO31 levels and FBXO46 degradation via a negative feedback loop. Collectively, our findings reveal that FBXO46 is a crucial proteasomal regulator of FBXO31 and thereby prevents senescence in normal growth conditions. They further indicate that FBXO46-mediated regulation of FBXO31 is abrogated following genotoxic stress to promote increased FBXO31 levels for maintenance of genomic stability.

Negative pressure pulmonary edema--a life-threatening condition in an eye care setting: a case report.

BACKGROUND: Negative pressure pulmonary edema is a potentially life-threatening complication after general anesthesia in young healthy individuals that results from upper airway obstruction followed by strong inspiratory effort. It is a known complication after nasal or upper airway surgery. Occurrence of such a life-threatening complication in an eye care setting where advanced intensive care is usually lacking is rare. CASE PRESENTATION: A 15-year-old Asian boy presented to our hospital with a penetrating eye injury caused by a pellet. Globe and vitreoretinal exploratory surgery was performed with the patient under general anesthesia. The patient's postoperative course was uneventful immediately after the procedure, but soon he developed negative pressure pulmonary edema. CONCLUSIONS: This case report highlights the importance of early diagnosis and prompt management of negative pressure pulmonary edema to save the life of the patient. Most ophthalmologic surgeries are performed with the patient under regional anesthesia; very few are done with the patient under general anesthesia. Intensive care facilities are needed in such settings for prompt management of such a serious and rare complication.

F-box protein FBXO31 directs degradation of MDM2 to facilitate p53-mediated growth arrest following genotoxic stress.

The tumor suppressor p53 plays a critical role in maintaining genomic stability. In response to genotoxic stress, p53 levels increase and induce cell-cycle arrest, senescence, or apoptosis, thereby preventing replication of damaged DNA. In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that directly interacts with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome. Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Because of the important role of MDM2 in modulating p53 function, it is critical to understand how MDM2 levels are regulated. Here we show that the F-box protein FBXO31, a candidate tumor suppressor encoded in 16q24.3 for which there is loss of heterozygosity in various solid tumors, is responsible for promoting MDM2 degradation. Following genotoxic stress, FBXO31 is phosphorylated by the DNA damage serine/threonine kinase ATM, resulting in increased levels of FBXO31. FBXO31 then interacts with and directs the degradation of MDM2, which is dependent on phosphorylation of MDM2 by ATM. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage.