UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors.

Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.

The Ubiquitin E3/E4 Ligase UBE4A Adjusts Protein Ubiquitylation and Accumulation at Sites of DNA Damage, Facilitating Double-Strand Break Repair.

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A's recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end resection at DSBs, and its abrogation leads to upregulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning the complex DDR network for accurate and balanced execution of DSB repair.

Weaning of Severe COVID-19 Mechanically Ventilated Patients: Experience within a Dedicated Unit in Israel.

BACKGROUND: Patients diagnosed with coronavirus disease-19 (COVID-19) who deteriorate to respiratory failure and require mechanical ventilation may later need to be weaned from the ventilator and undergo a rehabilitation process. The rate of weaning COVID-19 patients from mechanical ventilation is unknown. OBJECTIVES: To present our experience with ventilator weaning of COVID-19 patients in a dedicated facility. METHODS: A retrospective cohort study was conducted of 18 patients hospitalized in a COVID-19 dedicated ventilator weaning unit. RESULTS: Eighteen patients were hospitalized in the dedicated unit between 6 April and 19 May 2020. Of these, 88% (16/18) were weaned and underwent decannulation, while two patients deteriorated and were re-admitted to the intensive care unit. The average number of days spent in our department was 12. There was no statistically significant correlation between patient characteristics and time to weaning from ventilation or with the time to decannulation. CONCLUSIONS: Despite the high mortality of COVID-19 patients who require mechanical ventilation, most of the patients in our cohort were weaned in a relatively short period of time. Further large-scale studies are necessary to assess the cost effectiveness of dedicated COVID-19 departments for ventilator weaning.

ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair.

The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.

Pleuritis due to Mycobacterium xenopi without pulmonary infection.

Nontuberculous mycobacteria (NTM) may cause pulmonary and extra-pulmonary disease in both immunocompetent and immunocompromised patients. Pleuritis is an uncommon manifestation on NTM disease, and pleuritis caused by Mycobacterium xenopi has only been described once before. Because it is considered to be an environmental contaminant, isolation of M. xenopi from bronchopulmonary secretions or other sites is often dismissed. The disease caused by M. xenopi is usually a pulmonary infection and typically occurs in severely immunocompromised individuals or in immunocompetent patients with an underlying chronic lung disease. We describe an unusual case of pleuritis caused by M. xenopi in a patient without an underlying chronic lung disease and with no evidence of a concurrent M. xenopi pulmonary infection.

Nontuberculous mycobacteria infections of the pleura: A systematic review.

BACKGROUND: Nontuberculous mycobacterial (NTM) pleuritis is an uncommon manifestation of NTM infection. Case reports and small case series have shown a variable clinical course and high mortality rates. OBJECTIVE: To describe patients' characteristics, clinical presentation and outcomes of NTM pleural infections. METHODS: A systematic review of cases of NTM pleural infections published in PubMed-indexed journals from 1980 to 2021. RESULTS: A total of 206 cases of NTM pleural infections were found and analyzed. Fifty-eight percent of cases were males. The mean age was 57.5 yrs (range 9-87 yrs). Forty-three percent of patients were immunosuppressed, and 43% had a chronic lung disease; thirty-two percent had neither risk factor. In addition to the pleural infection, 67% of cases had a concurrent pulmonary NTM infection, and in 18 cases there was another extrapulmonary site of NTM infection. In 29% of cases the pleural infection was the sole manifestation of NTM disease. The most common isolated mycobacterium was Mycobacterium avium complex (65%). Fifty-three percent and 26% of patients required pleural effusion drainage and a surgical intervention, respectively, to treat the infection, in addition to anti-NTM chemotherapy. Forty percent of patients developed pneumothorax, 16% suffered from empyema, and 16.5% had broncho-pleural fistula. The reported mortality rate was 24%. CONCLUSION: NTM pleural infections may arise in immunocompetent and immunosuppressed patients, with or without chronic lung disease or concurrent NTM pulmonary infection. These infections carry a poor prognosis and a high risk of complications requiring surgical interventions in addition to anti-NTM chemotherapy.

Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress.

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds alpha-tubulin and promotes alpha/beta dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds alpha-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Delta mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Delta mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.

Mechanical Ventilation with Room Air is Feasible in a Moderate Acute Respiratory Distress Syndrome Pig Model - Implications for Disaster Situations and Low-Income Nations.

INTRODUCTION: Patients with respiratory failure are usually mechanically ventilated, mostly with fraction of inspired oxygen (FiO2) > 0.21. Minimizing FiO2 is increasingly an accepted standard. In underserved nations and disasters, salvageable patients requiring mechanical ventilation may outstrip oxygen supplies. STUDY OBJECTIVE: The hypothesis of the present study was that mechanical ventilation with FiO2 = 0.21 is feasible. This assumption was tested in an Acute Respiratory Distress Syndrome (ARDS) model in pigs. METHODS: Seventeen pigs were anesthetized, intubated, and mechanically ventilated with FiO2 = 0.4 and Positive End Expiratory Pressure (PEEP) of 5cmH2O. Acute Respiratory Distress Syndrome was induced by intravenous (IV) oleic acid (OA) infusion, and FiO2 was reduced to 0.21 after 45 minutes of stable moderate ARDS. If peripheral capillary oxygen saturation (SpO2) decreased below 80%, PEEP was increased gradually until maximum 20cmH2O, then inspiratory time elevated from one second to 1.4 seconds. RESULTS: Animals developed moderate ARDS (mean partial pressure of oxygen [PaO2]/FiO2 = 162.8, peak and mean inspiratory pressures doubled, and lung compliance decreased). The SpO2 decreased to <80% rapidly after FiO2 was decreased to 0.21. In 14/17 animals, increasing PEEP sufficed to maintain SpO2 > 80%. Only in 3/17 animals, elevation of FiO2 to 0.25 after PEEP reached 20cmH2O was needed to maintain SpO2 > 80%. Animals remained hemodynamically stable until euthanasia one hour later. CONCLUSIONS: In a pig model of moderate ARDS, mechanical ventilation with room air was feasible in 14/17 animals by elevating PEEP. These results in animal model support the potential feasibility of lowering FiO2 to 0.21 in some ARDS patients. The present study was conceived to address the ethical and practical paradigm of mechanical ventilation in disasters and underserved areas, which assumes that oxygen is mandatory in respiratory failure and is therefore a rate-limiting factor in care capacity allocation. Further studies are needed before paradigm changes are considered.

Nuclear export of Ho endonuclease of yeast via Msn5.

Exportin-5, an evolutionarily conserved nuclear export factor of the beta-karyopherin family, exports phosphorylated proteins and small noncoding RNAs. Msn5, the yeast ortholog, exports primarily phosphorylated cargoes including Ho endonuclease and a number of transcription factors and regulatory proteins. The Msn5-mediated nuclear export of Ho is dependent on phosphorylation of Thr225 by kinases of the DNA damage response pathway. Although Msn5 has been the object of many studies, no NES sequence capable of binding the exportin and/or of leading to Msn5-dependent export of a heterologous protein has been identified. Here we report identification of a 13-residue Ho sequence that interacts with Msn5 in vitro and directs Msn5-dependent nuclear export of GFP in vivo. A single point mutation in this 13-mer Ho NES abrogates both interaction with Msn5 and nuclear export of Ho and of GFP. However, this mutation, or of T225A, both of which abrogate nuclear export of Ho, does not interfere with its interaction with Msn5 implying that the exportin makes multiple contacts with its cargo. This can explain the lack of a conserved NES in Msn5 cargoes. Our results identify essential criteria for Msn5-mediated nuclear export of Ho: phosphorylation on HoT225, and interaction with the 13-mer Ho NES sequence.

New interacting partners of the F-box protein Ufo1 of yeast.

The yeast F-box protein Ufo1 recruits proteins for ubiquitylation by the SCF ubiquitin ligase complex preparing them for proteasomal degradation. Ufo1 has a role in maintenance of genome stability; its substrates include Ho endonuclease and Rad30 polymerase of error-prone DNA repair. Ufo1 is an unusual F-box protein, as it has three ubiquitin interacting motifs (UIMs). Deletion of the genomic UIMs is lethal; ectopic expression of UFO1 Delta UIMs extends protein half-life and arrests the cell cycle. A whole-genome study employing a TAP tag fused to the C-terminal UIMs did not identify Ufo1-interacting proteins. Here we therefore used stabilized N-terminally tagged Ufo1 Delta UIM as a strategy to identify Ufo1-interacting proteins by mass spectroscopy. We identified proteins that function in transcription, and an indirect interaction with Hsp70 molecular chaperones via the Skp1 adaptor; we also show that Ufo1 interacts with the 19S regulatory particle of the proteasome. Thus, our data augment the current network of known Ufo1 interacting proteins. We show directly that the UIMs are crucial for Ufo1 ubiquitylation in vivo, indicating that they facilitate turnover of SCF Ufo1 complexes. This allows recycling of the core subunits of the SCF complex and cell cycle progression.