Small molecules that disrupt RAD54-BLM interaction hamper tumor proliferation in colon cancer chemoresistance models.

RAD54 and BLM helicase play pivotal roles during homologous recombination repair (HRR) to ensure genome maintenance. BLM amino acids (aa 181-212) interact with RAD54 and enhance its chromatin remodeling activity. Functionally, this interaction heightens HRR, leading to a decrease in residual DNA damage in colon cancer cells. This contributes to chemoresistance in colon cancer cells against cisplatin, camptothecin, and oxaliplatin, eventually promoting tumorigenesis in preclinical colon cancer mouse models. ChIP-Seq analysis and validation revealed increased BLM and RAD54 corecruitment on the MRP2 promoter in camptothecin-resistant colon cancer cells, leading to BLM-dependent enhancement of RAD54-mediated chromatin remodeling. We screened the Prestwick small-molecule library, with the intent to revert camptothecin- and oxaliplatin-induced chemoresistance by disrupting the RAD54-BLM interaction. Three FDA/European Medicines Agency-approved candidates were identified that could disrupt this interaction. These drugs bound to RAD54, altered its conformation, and abrogated RAD54-BLM-dependent chromatin remodeling on G5E4 and MRP2 arrays. Notably, the small molecules also reduced HRR efficiency in resistant lines, diminished anchorage-independent growth, and hampered the proliferation of tumors generated using camptothecin- and oxaliplatin-resistant colon cancer cells in both xenograft and syngeneic mouse models in BLM-dependent manner. Therefore, the 3 identified small molecules can serve as possible viable candidates for adjunct therapy in colon cancer treatment.

Epidemiology and zoonotic potential of Livestock-associated Staphylococcus aureus isolated at Tamil Nadu, India.

BACKGROUND: Staphylococcus aureus is part of normal flora and also an opportunistic pathogen responsible for a wide range of infections in both humans and animals. Livestock-associated S. aureus (LA-SA) has gained importance in recent years due to its increased prevalence in recent years, becoming a worry in public health view. This study aimed to study the epidemiology of LA-SA strains in Madurai district, Tamil Nadu, India. METHODS: A total of 255 samples were collected from bovine and other small ruminants like goats and sheep nares (n = 129 and n = 126 respectively). Nasal swab samples were collected from study animals with sterile sample collecting cotton swabs (Hi-Media, Mumbai). Samples were transported to the lab in Cary-Blair Transport media for further analysis. The samples were tested for S. aureus using antibiotic selection and PCR-based assays. The pathogenicity of the bacteria was assessed using chicken embryo models and liver cross-sections were used for histopathology studies. RESULTS: The prevalence rate in bovine-associated samples was 42.63% but relatively low in the case of small ruminants associated samples with 28.57% only. The overall prevalence of S. aureus is found to 35.6% and MRSA 10.98% among the study samples. The antibiogram results that LA-SA isolates were susceptible to aminoglycosides and tetracyclines but resistant to ß-lactam drugs. The biofilm formation results showed that the LA-SA isolates are weak to high-capacity biofilm formers. The enterotoxigenic patterns revealed that most of the isolated strains are enterotoxigenic and possess classical enterotoxins. The survival analysis of chicken embryos suggested that the Bovine-associated strains were moderately pathogenic. CONCLUSION: The study concluded that economically important livestock animals can act as reservoirs for multi-drug resistant and pathogenic which in-turn is a concern for public health as well as livestock health.

Protocol for establishing a model for integrated influenza surveillance in Tamil Nadu, India.

The potential for influenza viruses to cause public health emergencies is great. The World Health Organisation (WHO) in 2005 concluded that the world was unprepared to respond to an influenza pandemic. Available surveillance guidelines for pandemic influenza lack the specificity that would enable many countries to establish operational surveillance plans. A well-designed epidemiological and virological surveillance is required to strengthen a country's capacity for seasonal, novel, and pandemic influenza detection and prevention. Here, we describe the protocol to establish a novel mechanism for influenza and SARS-CoV-2 surveillance in the four identified districts of Tamil Nadu, India. This project will be carried out as an implementation research. Each district will identify one medical college and two primary health centres (PHCs) as sentinel sites for collecting severe acute respiratory infections (SARI) and influenza like illness (ILI) related information, respectively. For virological testing, 15 ILI and 10 SARI cases will be sampled and tested for influenza A, influenza B, and SARS-CoV-2 every week. Situation analysis using the WHO situation analysis tool will be done to identify the gaps and needs in the existing surveillance systems. Training for staff involved in disease surveillance will be given periodically. To enhance the reporting of ILI/SARI for sentinel surveillance, trained project staff will collect information from all ILI/SARI patients attending the sentinel sites using pre-tested tools. Using time, place, and person analysis, alerts for abnormal increases in cases will be generated and communicated to health authorities to initiate response activities. Advanced epidemiological analysis will be used to model influenza trends over time. Integrating virological and epidemiological surveillance data with advanced analysis and timely communication can enhance local preparedness for public health emergencies. Good quality surveillance data will facilitate an understanding outbreak severity and disease seasonality. Real-time data will help provide early warning signals for prevention and control of influenza and COVID-19 outbreaks. The implementation strategies found to be effective in this project can be scaled up to other parts of the country for replication and integration.

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.

Enhancement of c-Myc degradation by BLM helicase leads to delayed tumor initiation.

The spectrum of tumors that arise owing to the overexpression of c-Myc and loss of BLM is very similar. Hence, it was hypothesized that the presence of BLM negatively regulates c-Myc functions. By using multiple isogenic cell lines, we observed that the decrease of endogenous c-Myc levels that occurs in the presence of BLM is reversed when the cells are treated with proteasome inhibitors, indicating that BLM enhances c-Myc turnover. Whereas the N-terminal region of BLM interacts with c-Myc, the rest of the helicase interacts with the c-Myc E3 ligase Fbw7. The two BLM domains act as 'clamp and/or adaptor', enhancing the binding of c-Myc to Fbw7. BLM promotes Fbw7-dependent K48-linked c-Myc ubiquitylation and its subsequent degradation in a helicase-independent manner. A subset of BLM-regulated genes that are also targets of c-Myc were determined and validated at both RNA and protein levels. To obtain an in vivo validation of the effect of BLM on c-Myc-mediated tumor initiation, isogenic cells from colon cancer cells that either do or do not express BLM had been manipulated to block c-Myc expression in a controlled manner. By using these cell lines, the metastatic potential and rate of initiation of tumors in nude mice were determined. The presence of BLM decreases c-Myc-mediated invasiveness and delays tumor initiation in a mouse xenograft model. Consequently, in tumors that express BLM but not c-Myc, we observed a decreased ratio of proliferation to apoptosis together with a suppressed expression of the angiogenesis marker CD31. Hence, partly owing to its regulation of c-Myc stability, BLM acts as a 'caretaker tumor suppressor'.

Ubiquitin-dependent recruitment of the Bloom syndrome helicase upon replication stress is required to suppress homologous recombination.

Limiting the levels of homologous recombination (HR) that occur at sites of DNA damage is a major role of BLM helicase. However, very little is known about the mechanisms dictating its relocalization to these sites. Here, we demonstrate that the ubiquitin/SUMO-dependent DNA damage response (UbS-DDR), controlled by the E3 ligases RNF8/RNF168, triggers BLM recruitment to sites of replication fork stalling via ubiquitylation in the N-terminal region of BLM and subsequent BLM binding to the ubiquitin-interacting motifs of RAP80. Furthermore, we show that this mechanism of BLM relocalization is essential for BLM's ability to suppress excessive/uncontrolled HR at stalled replication forks. Unexpectedly, we also uncovered a requirement for RNF8-dependent ubiquitylation of BLM and PML for maintaining the integrity of PML-associated nuclear bodies and as a consequence the localization of BLM to these structures. Lastly, we identified a novel role for RAP80 in preventing proteasomal degradation of BLM in unstressed cells. Taken together, these data highlight an important biochemical link between the UbS-DDR and BLM-dependent pathways involved in maintaining genome stability.