"I feel drug resistance testing allowed us to make an informed decision": qualitative insights on the role of HIV drug resistance mutation testing among children and pregnant women living with HIV in western Kenya.

BACKGROUND: Pregnant women and children living with HIV in Kenya achieve viral suppression (VS) at lower rates than other adults. While many factors contribute to these low rates, the acquisition and development of HIV drug resistance mutations (DRMs) are a contributing factor. Recognizing the significance of DRMs in treatment decisions, resource-limited settings are scaling up national DRM testing programs. From provider and patient perspectives, however, optimal ways to operationalize and scale-up DRM testing in such settings remain unclear. METHODS: Our mixed methods study evaluates the attitudes towards, facilitators to, and barriers to DRM testing approaches among children and pregnant women on antiretroviral therapy (ART) in five HIV treatment facilities in Kenya. We conducted 68 key informant interviews (KIIs) from December 2019 to December 2020 with adolescents, caregivers, pregnant women newly initiating ART or with a high viral load, and providers, laboratory/facility leadership, and policy makers. Our KII guides covered the following domains: (1) DRM testing experiences in routine care and through our intervention and (2) barriers and facilitators to routine and point-of-care DRM testing scale-up. We used inductive coding and thematic analysis to identify dominant themes with convergent and divergent subthemes. RESULTS: The following themes emerged from our analysis: (1) DRM testing and counseling were valuable to clinical decision-making and reassuring to patients, with timely results allowing providers to change patient ART regimens faster; (2) providers and policymakers desired an amended and potentially decentralized DRM testing process that incorporates quicker sample-to-results turn-around-time, less burdensome procedures, and greater patient and provider "empowerment" to increase comfort with testing protocols; (3) facility-level delays, deriving from overworked facilities and sample tracking difficulties, were highlighted as areas for improvement. CONCLUSIONS: DRM testing has the potential to considerably improve patient health outcomes. Key informants recognized several obstacles to implementation and desired a more simplified, time-efficient, and potentially decentralized DRM testing process that builds provider comfort and confidence with DRM testing protocols. Further investigating the implementation, endurance, and effectiveness of DRM testing training is critical to addressing the barriers and areas of improvement highlighted in our study. TRIAL REGISTRATION: NCT03820323.

A CRISPR-Cas9 screen identifies EXO1 as a formaldehyde resistance gene.

Fanconi Anemia (FA) is a rare, genome instability-associated disease characterized by a deficiency in repairing DNA crosslinks, which are known to perturb several cellular processes, including DNA transcription, replication, and repair. Formaldehyde, a by-product of metabolism, is thought to drive FA by generating DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs). However, the impact of formaldehyde on global cellular pathways has not been investigated thoroughly. Herein, using a pangenomic CRISPR-Cas9 screen, we identify EXO1 as a critical regulator of formaldehyde-induced DNA lesions. We show that EXO1 knockout cell lines exhibit formaldehyde sensitivity leading to the accumulation of replicative stress, DNA double-strand breaks, and quadriradial chromosomes, a typical feature of FA. After formaldehyde exposure, EXO1 is recruited to chromatin, protects DNA replication forks from degradation, and functions in parallel with the FA pathway to promote cell survival. In vitro, EXO1-mediated exonuclease activity is proficient in removing DPCs. Collectively, we show that EXO1 limits replication stress and DNA damage to counteract formaldehyde-induced genome instability.

A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response.

Helix-destabilizing DNA lesions induced by environmental mutagens such as UV light cause genomic instability by strongly blocking the progression of DNA replication forks (RFs). At blocked RF, single-stranded DNA (ssDNA) accumulates and is rapidly bound by Replication Protein A (RPA) complexes. Such stretches of RPA-ssDNA constitute platforms for recruitment/activation of critical factors that promote DNA synthesis restart. However, during periods of severe replicative stress, RPA availability may become limiting due to inordinate sequestration of this multifunctional complex on ssDNA, thereby negatively impacting multiple vital RPA-dependent processes. Here, we performed a genome-wide screen to identify factors that restrict the accumulation of RPA-ssDNA during UV-induced replicative stress. While this approach revealed some expected "hits" acting in pathways such as nucleotide excision repair, translesion DNA synthesis, and the intra-S phase checkpoint, it also identified SCAI, whose role in the replicative stress response was previously unappreciated. Upon UV exposure, SCAI knock-down caused elevated accumulation of RPA-ssDNA during S phase, accompanied by reduced cell survival and compromised RF progression. These effects were independent of the previously reported role of SCAI in 53BP1-dependent DNA double-strand break repair. We also found that SCAI is recruited to UV-damaged chromatin and that its depletion promotes nascent DNA degradation at stalled RF. Finally, we (i) provide evidence that EXO1 is the major nuclease underlying ssDNA formation and DNA replication defects in SCAI knockout cells and, consistent with this, (ii) demonstrate that SCAI inhibits EXO1 activity on a ssDNA gap in vitro. Taken together, our data establish SCAI as a novel regulator of the UV-induced replicative stress response in human cells.

A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene.

BACKGROUND: Familial ovarian cancer (OC) cases not harbouring pathogenic variants in either of the BRCA1 and BRCA2 OC-predisposing genes, which function in homologous recombination (HR) of DNA, could involve pathogenic variants in other DNA repair pathway genes. METHODS: Whole exome sequencing was used to identify rare variants in HR genes in a BRCA1 and BRCA2 pathogenic variant negative OC family of French Canadian (FC) ancestry, a population exhibiting genetic drift. OC cases and cancer-free individuals from FC and non-FC populations were investigated for carrier frequency of FANCI c.1813C>T; p.L605F, the top-ranking candidate. Gene and protein expression were investigated in cancer cell lines and tissue microarrays, respectively. RESULTS: In FC subjects, c.1813C>T was more common in familial (7.1%, 3/42) than sporadic (1.6%, 7/439) OC cases (P = 0.048). Carriers were detected in 2.5% (74/2950) of cancer-free females though female/male carriers were more likely to have a first-degree relative with OC (121/5249, 2.3%; Spearman correlation = 0.037; P = 0.011), suggesting a role in risk. Many of the cancer-free females had host factors known to reduce risk to OC which could influence cancer risk in this population. There was an increased carrier frequency of FANCI c.1813C>T in BRCA1 and BRCA2 pathogenic variant negative OC families, when including the discovery family, compared to cancer-free females (3/23, 13%; OR = 5.8; 95%CI = 1.7-19; P = 0.005). In non-FC subjects, 10 candidate FANCI variants were identified in 4.1% (21/516) of Australian OC cases negative for pathogenic variants in BRCA1 and BRCA2, including 10 carriers of FANCI c.1813C>T. Candidate variants were significantly more common in familial OC than in sporadic OC (P = 0.04). Localization of FANCD2, part of the FANCI-FANCD2 (ID2) binding complex in the Fanconi anaemia (FA) pathway, to sites of induced DNA damage was severely impeded in cells expressing the p.L605F isoform. This isoform was expressed at a reduced level, destabilized by DNA damaging agent treatment in both HeLa and OC cell lines, and exhibited sensitivity to cisplatin but not to a poly (ADP-ribose) polymerase inhibitor. By tissue microarray analyses, FANCI protein was consistently expressed in fallopian tube epithelial cells and only expressed at low-to-moderate levels in 88% (83/94) of OC samples. CONCLUSIONS: This is the first study to describe candidate OC variants in FANCI, a member of the ID2 complex of the FA DNA repair pathway. Our data suggest that pathogenic FANCI variants may modify OC risk in cancer families.

Animal models of Fanconi anemia: A developmental and therapeutic perspective on a multifaceted disease.

Fanconi anemia (FA) is a genetic disorder characterized by developmental abnormalities, progressive bone marrow failure, and increased susceptibility to cancer. FA animal models have been useful to understand the pathogenesis of the disease. Herein, we review FA developmental models that have been developed to simulate human FA, focusing on zebrafish and mouse models. We summarize the recapitulated phenotypes observed in these in vivo models including bone, gametogenesis and sterility defects, as well as marrow failure. We also discuss the relevance of aldehydes in pathogenesis of FA, emphasizing on hematopoietic defects. In addition, we provide a summary of potential therapeutic agents, such as aldehyde scavengers, TGFß inhibitors, and gene therapy for FA. The diversity of FA animal models makes them useful for understanding FA etiology and allows the discovery of new therapies.

Clinical characteristics of Coronavirus Disease 2019 and development of a prediction model for prolonged hospital length of stay.

BACKGROUND: The epidemic of Coronavirus Disease 2019 (COVID-19) has become a global health emergency, but the clinical characteristics of COVID-19 are not fully described. We aimed to describe the clinical characteristics of COVID-19 outside of Wuhan city; and to develop a multivariate model to predict the risk of prolonged length of stay in hospital (ProLOS). METHODS: The study was conducted in a tertiary care hospital in Zhejiang province from January to February 20, 2020. Medical records of all confirmed cases of COVID-19 were retrospectively reviewed. Patients were categorized into the ProLOS and non-ProLOS groups by hospital length of stay greater and less than 14 days, respectively. Conventional descriptive statistics were applied. Multivariate regression model was built to predict the risk of ProLOS, with variables selected using stepwise approach. RESULTS: A total of 75 patients with confirmed COVID-19 were included for quantitative analysis, including 25 (33%) patients in the ProLOS group. ProLOS patients were more likely to have history of traveling to Wuhan (68% vs. 28%; P=0.002). Patients in the ProLOS group showed lower neutrophil counts [median (IQR): 2.50 (1.77-3.23) ×109/L vs. 2.90 (2.21-4.19) ×109/L; P=0.048], higher partial thrombin time (PT) (13.42±0.63 vs. 13.10±0.48 s; P=0.029), lower D-Dimer [0.26 (0.22-0.46) vs. 0.44 (0.32-0.84) mg/L; P=0.012]. There was no patient died and no severe case in our cohort. The overall LOS was 11 days (IQR, 5-15 days). The median cost for a hospital stay was 7,388.19 RMB (IQR, 5,085.39-11,145.44). The prediction model included five variables of procalcitonin, heart rate, epidemiological history, lymphocyte count and cough. The discrimination of the model was 84.8% (95% CI: 75.3% to 94.4%). CONCLUSIONS: Our study described clinical characteristics of COVID-19 outside of Wuhan city and found that the illness was less severe than that in the core epidemic region. A multivariate model was developed to predict ProLOS, which showed good discrimination.

A Fanci knockout mouse model reveals common and distinct functions for FANCI and FANCD2.

Fanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci -/- mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci-/- Fancd2-/- also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.

The Tumor Suppressor PALB2: Inside Out.

Partner and Localizer of BRCA2 (PALB2) has emerged as an important and versatile player in genome integrity maintenance. Biallelic mutations in PALB2 cause Fanconi anemia (FA) subtype FA-N, whereas monoallelic mutations predispose to breast, and pancreatic familial cancers. Herein, we review recent developments in our understanding of the mechanisms of regulation of the tumor suppressor PALB2 and its functional domains. Regulation of PALB2 functions in DNA damage response and repair occurs on multiple levels, including homodimerization, phosphorylation, and ubiquitylation. With a molecular emphasis, we present PALB2-associated cancer mutations and their detailed analysis by functional assays.

Discovery of novel HSP90 inhibitors that induced apoptosis and impaired autophagic flux in A549 lung cancer cells.

Heat shock protein 90 (HSP90) inhibition has aroused increasing enthusiasm in antitumor strategies in recent years. According to our previous studies, we synthesized a series of coumarin pyrazoline compounds HCP1-HCP6 that might be HSP90 inhibitors. Interactions between HCP1-HCP6 and HSP90 were examined and antitumor activities of them were investigated in A549 lung cancer cells. Results showed that all the six derivatives could interact with HSP90, in which HCP1 exhibited the best binding ability and inhibited the activity of HSP90. Meanwhile, HCP1-HCP6 reduced the cell viability of A549 cells and HCP1 possessed the lowest IC50 value. Above all HCP1 exerted better HSP90 inhibitory and anticancer effects than our initially identified HSP90 inhibitor DPB. As to the underlying mechanism, HCP1-HCP6 not only induced apoptosis as DPB but also blocked autophagic flux in A549 cells. Therefore, we discovered a novel HSP90 inhibitor HCP1 that had better biological activity and provided us a useful tool to explore the underlying mechanism of lung cancer therapy.

A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1 S-glutathionylation and inducing apoptosis.

The level of glutathione (GSH) is increased in many cancer cells. Consuming intracellular GSH by chemical small molecules that specifically target GSH is a new strategy to treat cancer. Recently, we synthesized and proved that a new compound 2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)cyclohexa-2,5-diene-1,4-dione (PBQC) could target to and consume intracellular GSH specifically, but, it is not clear if PBQC can affect cancer cell growth and the activity of the nuclear factor-erythroid 2-related factor 2 (Nrf2) which is a key factor involved in regulation of cancer cell growth. In this study, we addressed these questions. We found that PBQC suppressed cancer cell growth through increasing the activity of Nrf2, while it did not inhibit normal vascular endothelial cell growth. Furthermore, we demonstrated that PBQC can cause Keap-1 protein S-glutathionylation and promote Nrf2 nuclear translocation as well as the expression of pro-apoptosis genes. As a result, the cancer cells underwent apoptosis. Here, we provide a new Nrf2 activator, PBQC that can promote the expressions of pro-apoptosis genes downstream Nrf2. The data suggest that PBQC is a potential lead-compound for development of new anti-cancer drugs.

Novel indolyl-chalcone derivatives inhibit A549 lung cancer cell growth through activating Nrf-2/HO-1 and inducing apoptosis in vitro and in vivo.

Increasing evidence indicates that Nrf-2, named the nuclear factor-erythroid 2-related factor, may perform anticancer function. In this study, a series of novel substituted phenyl- (3-methyl-1H-indol-2-yl)-prop-2-en-1-one (indolyl-chalcone) derivatives were synthesized and their effects on Nrf-2 activity were observed. We found that compounds 3a-3d and 6c elevated Nrf-2 activity. Then we evaluated their anticancer activities in vitro and in vivo by utilizing human lung cancer cell line A549. The in vitro results showed that among the compounds, 3d performed effectively anti-growth activity by inducing A549 lung cancer cell apoptosis and activating Nrf-2/HO-1 (heme oxygenase-1) pathway. In vivo, we proved that compound 3d inhibited the tumor growth effectively through inducing cell apoptosis without affecting CAM normal angiogenesis. These data suggest that our discovery of a novel Nrf-2 activator compound 3d would provide a new point of human lung cancer treatment.