A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

Cabergoline as a Novel Strategy for Post-Pregnancy Breast Cancer Prevention in Mice and Human.

Post-pregnancy breast cancer often carries a poor prognosis, posing a major clinical challenge. The increasing trend of later-life pregnancies exacerbates this risk, highlighting the need for effective chemoprevention strategies. Current options, limited to selective estrogen receptor modulators, aromatase inhibitors, or surgical procedures, offer limited efficacy and considerable side effects. Here, we report that cabergoline, a dopaminergic agonist, reduces the risk of breast cancer post-pregnancy in a Brca1/P53-deficient mouse model, with implications for human breast cancer prevention. We show that a single dose of cabergoline administered post-pregnancy significantly delayed the onset and reduced the incidence of breast cancer in Brca1/P53-deficient mice. Histological analysis revealed a notable acceleration in post-lactational involution over the short term, characterized by increased apoptosis and altered gene expression related to ion transport. Over the long term, histological changes in the mammary gland included a reduction in the ductal component, decreased epithelial proliferation, and a lower presence of recombinant Brca1/P53 target cells, which are precursors of tumors. These changes serve as indicators of reduced breast cancer susceptibility. Additionally, RNA sequencing identified gene expression alterations associated with decreased proliferation and mammary gland branching. Our findings highlight a mechanism wherein cabergoline enhances the protective effect of pregnancy against breast cancer by potentiating postlactational involution. Notably, a retrospective cohort study in women demonstrated a markedly lower incidence of post-pregnancy breast cancer in those treated with cabergoline compared to a control group. Our work underscores the importance of enhancing postlactational involution as a strategy for breast cancer prevention, and identifies cabergoline as a promising, low-risk option in breast cancer chemoprevention. This strategy has the potential to revolutionize breast cancer prevention approaches, particularly for women at increased risk due to genetic factors or delayed childbirth, and has wider implications beyond hereditary breast cancer cases.

Cost-effective complete genome sequencing using the MinION platform for identification of recombinant enteroviruses.

IMPORTANCE: By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study's findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.

A transcriptional response to replication stress selectively expands a subset of Brca2-mutant mammary epithelial cells.

Germline BRCA2 mutation carriers frequently develop luminal-like breast cancers, but it remains unclear how BRCA2 mutations affect mammary epithelial subpopulations. Here, we report that monoallelic Brca2mut/WT mammary organoids subjected to replication stress activate a transcriptional response that selectively expands Brca2mut/WT luminal cells lacking hormone receptor expression (HR-). While CyTOF analyses reveal comparable epithelial compositions among wildtype and Brca2mut/WT mammary glands, Brca2mut/WT HR- luminal cells exhibit greater organoid formation and preferentially survive and expand under replication stress. ScRNA-seq analysis corroborates the expansion of HR- luminal cells which express elevated transcript levels of Tetraspanin-8 (Tspan8) and Thrsp, plus pathways implicated in replication stress survival including Type I interferon responses. Notably, CRISPR/Cas9-mediated deletion of Tspan8 or Thrsp prevents Brca2mut/WT HR- luminal cell expansion. Our findings indicate that Brca2mut/WT cells activate a transcriptional response after replication stress that preferentially favours outgrowth of HR- luminal cells through the expression of interferon-responsive and mammary alveolar genes.

Substitution of Coxsackievirus A16 VP1 BC and EF Loop Altered the Protective Immune Responses in Chimera Enterovirus A71.

Hand, foot and mouth disease (HFMD) is a childhood disease caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). Capsid loops are important epitopes for EV-A71 and CV-A16. Seven chimeric EV-A71 (ChiE71) involving VP1 BC (45.5% similarity), DE, EF, GH and HI loops, VP2 EF loop and VP3 GH loop (91.3% similarity) were substituted with corresponding CV-A16 loops. Only ChiE71-1-BC, ChiE71-1-EF, ChiE71-1-GH and ChiE71-3-GH were viable. EV-A71 and CV-A16 antiserum neutralized ChiE71-1-BC and ChiE71-1-EF. Mice immunized with inactivated ChiE71 elicited high IgG, IFN-γ, IL-2, IL-4 and IL-10. Neonatal mice receiving passive transfer of WT EV-A71, ChiE71-1-EF and ChiE71-1-BC immune sera had 100%, 80.0% and no survival, respectively, against lethal challenges with EV-A71, suggesting that the substituted CV-A16 loops disrupted EV-A71 immunogenicity. Passive transfer of CV-A16, ChiE71-1-EF and ChiE71-1-BC immune sera provided 40.0%, 20.0% and 42.9% survival, respectively, against CV-A16. One-day-old neonatal mice immunized with WT EV-A71, ChiE71-1-BC, ChiE71-1-EF and CV-A16 achieved 62.5%, 60.0%, 57.1%, and no survival, respectively, after the EV-A71 challenge. Active immunization using CV-A16 provided full protection while WT EV-A71, ChiE71-1-BC and ChiE71-1-EF immunization showed partial cross-protection in CV-A16 lethal challenge with survival rates of 50.0%, 20.0% and 40%, respectively. Disruption of a capsid loop could affect virus immunogenicity, and future vaccine design should include conservation of the enterovirus capsid loops.

Development and user testing study of MozzHub: a bipartite network-based dengue hotspot detector.

Traditionally, dengue is controlled by fogging, and the prime location for the control measure is at the patient's residence. However, when Malaysia was hit by the first wave of the Coronavirus disease (COVID-19), and the government-imposed movement control order, dengue cases have decreased by more than 30% from the previous year. This implies that residential areas may not be the prime locations for dengue-infected mosquitoes. The existing early warning system was focused on temporal prediction wherein the lack of consideration for spatial component at the microlevel and human mobility were not considered. Thus, we developed MozzHub, which is a web-based application system based on the bipartite network-based dengue model that is focused on identifying the source of dengue infection at a small spatial level (400 m) by integrating human mobility and environmental predictors. The model was earlier developed and validated; therefore, this study presents the design and implementation of the MozzHub system and the results of a preliminary pilot test and user acceptance of MozzHub in six district health offices in Malaysia. It was found that the MozzHub system is well received by the sample of end-users as it was demonstrated as a useful (77.4%), easy-to-operate system (80.6%), and has achieved adequate client satisfaction for its use (74.2%).

SARS-CoV-2 genomic surveillance in Malaysia: displacement of B.1.617.2 with AY lineages as the dominant Delta variants and the introduction of Omicron during the fourth epidemic wave.

OBJECTIVES: This study reported SARS-CoV-2 whole genome sequencing results from June 2021 to January 2022 from seven genome sequencing centers in Malaysia as part of the national surveillance program. METHODS: COVID-19 samples that tested positive by reverse transcription polymerase chain reaction and with cycle threshold values <30 were obtained throughout Malaysia. Sequencing of SARS-CoV-2 complete genomes was performed using Illumina, Oxford Nanopore, or Ion Torrent platforms. A total of 6163 SARS-CoV-2 complete genome sequences were generated over the surveillance period. All sequences were submitted to the Global Initiative on Sharing All Influenza Data database. RESULTS: From June 2021 to January 2022, Malaysia experienced the fourth wave of COVID-19 dominated by the Delta variant of concern, including the original B.1.617.2 lineage and descendant AY lineages. The B.1.617.2 lineage was identified as the early dominant circulating strain throughout the country but over time, was displaced by AY.59 and AY.79 lineages in Peninsular (west) Malaysia, and the AY.23 lineage in east Malaysia. In December 2021, pilgrims returning from Saudi Arabia facilitated the introduction and spread of the BA.1 lineage (Omicron variant of concern) in the country. CONCLUSION: The changing trends of circulating SARS-CoV-2 lineages were identified, with differences observed between west and east Malaysia. This initiative highlighted the importance of leveraging research expertise in the country to facilitate pandemic response and preparedness.

Rats and the city: Implications of urbanization on zoonotic disease risk in Southeast Asia.

Urbanization is rapidly transforming much of Southeast Asia, altering the structure and function of the landscape, as well as the frequency and intensity of the interactions between people, animals, and the environment. In this study, we explored the impact of urbanization on zoonotic disease risk by simultaneously characterizing changes in the ecology of animal reservoirs (rodents), ectoparasite vectors (ticks), and pathogens across a gradient of urbanization in Kuching, a city in Malaysian Borneo. We sampled 863 rodents across rural, developing, and urban locations and found that rodent species diversity decreased with increasing urbanization-from 10 species in the rural location to 4 in the rural location. Notably, two species appeared to thrive in urban areas, as follows: the invasive urban exploiter Rattus rattus (n = 375) and the native urban adapter Sundamys muelleri (n = 331). R. rattus was strongly associated with built infrastructure across the gradient and carried a high diversity of pathogens, including multihost zoonoses capable of environmental transmission (e.g., Leptospira spp.). In contrast, S. muelleri was restricted to green patches where it was found at high densities and was strongly associated with the presence of ticks, including the medically important genera Amblyomma, Haemaphysalis, and Ixodes. Our analyses reveal that zoonotic disease risk is elevated and heterogeneously distributed in urban environments and highlight the potential for targeted risk reduction through pest management and public health messaging.

Early Neutrophilia Marked by Aerobic Glycolysis Sustains Host Metabolism and Delays Cancer Cachexia.

An elevated neutrophil-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, using murine model systems of colorectal and pancreatic cancer we show that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, similar to cancer cells. Although pharmacological inhibition of aerobic glycolysis slows down tumor growth in C26 tumor-bearing mice, it precipitates cachexia, thereby shortening the overall survival. This negative effect may be explained by our observation that acute depletion of neutrophils in pre-cachectic mice impairs systemic glucose homeostasis secondary to altered hepatic lipid processing. Thus, changes in neutrophil number, distribution, and metabolism play an adaptive role in host metabolic homeostasis during cancer progression. Our findings provide insight into early events during cancer progression to cachexia, with implications for therapy.

Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection.

Hand, foot and mouth disease (HFMD) caused by Human Enterovirus A71 (HEVA71) infection is typically a benign infection. However, in minority of cases, children can develop severe neuropathology that culminate in fatality. Approximately 36.9% of HEVA71-related hospitalizations develop neurological complications, of which 10.5% are fatal. Yet, the mechanism by which HEVA71 induces these neurological deficits remain unclear. Here, we show that HEVA71-infected astrocytes release CXCL1 which supports viral replication in neurons by activating the CXCR2 receptor-associated ERK1/2 signaling pathway. Elevated CXCL1 levels correlates with disease severity in a HEVA71-infected mice model. In humans infected with HEVA71, high CXCL1 levels are only present in patients presenting neurological complications. CXCL1 release is specifically triggered by VP4 synthesis in HEVA71-infected astrocytes, which then acts via its receptor CXCR2 to enhance viral replication in neurons. Perturbing CXCL1 signaling or VP4 myristylation strongly attenuates viral replication. Treatment with AZD5069, a CXCL1-specific competitor, improves survival and lessens disease severity in infected animals. Collectively, these results highlight the CXCL1-CXCR2 signaling pathway as a potential target against HFMD neuropathogenesis.

Changing predominant SARS-CoV-2 lineages drives successive COVID-19 waves in Malaysia, February 2020 to March 2021.

Malaysia has experienced three waves of coronavirus disease 2019 (COVID-19) as of March 31, 2021. We studied the associated molecular epidemiology and SARS-CoV-2 seroprevalence during the third wave. We obtained 60 whole-genome SARS-CoV-2 sequences between October 2020 and January 2021 in Kuala Lumpur/Selangor and analyzed 989 available Malaysian sequences. We tested 653 residual serum samples collected between December 2020 to April 2021 for anti-SARS-CoV-2 total antibodies, as a proxy for population immunity. The first wave (January 2020) comprised sporadic imported cases from China of early Pango lineages A and B. The second wave (March-June 2020) was associated with lineage B.6. The ongoing third wave (from September 2020) was propagated by a state election in Sabah. It is due to lineage B.1.524 viruses containing spike mutations D614G and A701V. Lineages B.1.459, B.1.470, and B.1.466.2 were likely imported from the region and confined to Sarawak state. Direct age-standardized seroprevalence in Kuala Lumpur/Selangor was 3.0%. The second and third waves were driven by super-spreading events and different circulating lineages. Malaysia is highly susceptible to further waves, especially as alpha (B.1.1.7) and beta (B.1.351) variants of concern were first detected in December 2020/January 2021. Increased genomic surveillance is critical.

Inhibitors of the Bub1 spindle assembly checkpoint kinase: synthesis of BAY-320 and comparison with 2OH-BNPP1.

Bub1 is a serine/threonine kinase proposed to function centrally in mitotic chromosome alignment and the spindle assembly checkpoint (SAC); however, its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors are invaluable tools for investigating kinase function, we evaluated two potential Bub1 inhibitors: 2OH-BNPPI and BAY-320. After confirming that both inhibit Bub1 in vitro, we developed a cell-based assay for Bub1 inhibition. We overexpressed a fusion of Histone 2B and Bub1 kinase region, tethering it in proximity to H2A to generate a strong ectopic H2ApT120 signal along chromosome arms. Ectopic signal was effectively inhibited by BAY-320, but not 2OH-BNPP1 at concentrations tested. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localization. Preliminary experiments using BAY-320 suggest a minor role for Bub1 kinase activity in chromosome alignment and the SAC; however, BAY-320 may exhibit off-target effects at the concentration required. Thus, 2OH-BNPP1 may not be an effective Bub1 inhibitor in cellulo, and while BAY-320 can inhibit Bub1 in cells, off-target effects highlight the need for improved Bub1 inhibitors.

First detection of a novel 'unknown host' flavivirus in a Malaysian rodent.

Current phylogenetic analysis of the flavivirus genus has identified a group of mosquito-borne viruses for which the vertebrate hosts are currently unknown. Here we report the identification of a novel member of this group from a peridomestic rodent species (Sundamys muelleri) collected in Sarawak, Malaysia in 2016. We propose to name this novel flavivirus Batu Kawa virus after the location in which it was identified, with the abbreviation BKWV. Characterization of the BKWV genome allowed identification of putative mature peptides, potential enzyme motifs and conserved structural elements. Phylogenetic analysis found BKWV to be most closely related to Nhumirim virus (from Brazil) and Barkedji virus (from Senegal and Israel). Both of these viruses have been identified in Culex mosquitoes and belong to a group of viruses with unknown vertebrate hosts. This is the first known report of a member of this group of viruses from a potential mammalian host.

A pan-coronavirus RT-PCR assay for rapid viral screening of animal, human, and environmental specimens.

We examined a collection of 386 animal, 451 human, and 109 archived bioaerosol samples with a new pan-species coronavirus molecular assay. Thirty-eight (4.02%) of 946 specimens yielded evidence of human or animal coronaviruses. Our findings demonstrate the utility of employing the pan-CoV RT-PCR assay in detecting varied coronavirus among human, animal, and environmental specimens. This RT-PCR assay might be employed as a screening diagnostic for early detection of coronaviruses incursions or prepandemic coronavirus emergence in animal or human populations.

Shifts in mosquito diversity and abundance along a gradient from oil palm plantations to conterminous forests in Borneo.

Deforestation precipitates spillover of enzootic, vector-borne viruses into humans, but specific mechanisms for this effect have rarely been investigated. Expansion of oil palm cultivation is a major driver of deforestation. Here, we demonstrate that mosquito abundance decreased over ten stepwise distances from interior forest into conterminous palm plantations in Borneo. Diversity in interior plantation narrowed to one species, Aedes albopictus, a potential bridge vector for spillover of multiple viruses. A. albopictus was equally abundant across all distances in forests, forest-plantation edge, and plantations, while A. niveus, a known vector of sylvatic dengue virus, was found only in forests. A. albopictus collections were significantly female-biased in plantation but not in edge or forest. Our data reveal that the likelihood of encountering any mosquito is greater in interior forest and edge than plantation, while the likelihood of encountering A. albopictus is equivalent across the gradient sampled from interior plantation to interior forest.

A novel orally infected hamster model for Coxsackievirus A16 hand-foot-and-mouth disease and encephalomyelitis.

Coxsackievirus A16 (CV-A16) is one of the major causes of mild and self-limiting hand-foot-and-mouth disease (HFMD) in young children, which may occasionally leads to serious neurological complications. In this study, we had developed a novel, consistent, orally infected CV-A16 HFMD hamster model with encephalomyelitis. Four groups of 7-day-old hamsters in a kinetic study were orally infected with mouse-adapted CV-A16 strains and sacrificed at 1-4 days post infection (dpi), respectively. Tissues were studied by light microscopy, immunohistochemistry to detect viral antigens, in situ hybridization to detect viral RNA, and by viral titration. In a separate transmission experiment, orally infected index hamsters were housed together with contact hamsters to investigate oral and fecal viral shedding by virus culture and reverse transcription polymerase chain reaction (RT-PCR). At severe infection/death endpoints, index and contact hamster infection were also histopathologically analyzed. In the kinetic study, infected hamsters developed signs of infection at 4 dpi. Viral antigens/RNA were localized to brainstem (medulla/pons; reticular formation and motor trigeminal nucleus) and spinal cord anterior horn neurons, oral squamous epithelia and epidermis from 3 to 4 dpi. Salivary and lacrimal glands, myocardium, brown adipose tissue, intestinal smooth muscle, and skeletal muscle infection was also demonstrated. Viremia at 1 dpi and increasing viral titers in various tissues were observed from 2 dpi. In the transmission study, all contact hamsters developed disease 3-5 days later than index hamsters, but demonstrated similar histopathological findings at endpoint. Viral culture and RT-PCR positive oral washes and feces confirmed viral shedding. Our hamster model, orally infected by the natural route for human infection, confirmed CV-A16 neurotropism and demonstrated squamous epitheliotropism reminiscent of HFMD, attributes not found in other animal models. It should be useful to investigate neuropathogenesis, model person-to-person transmission, and for testing antiviral drugs and vaccines.

Author Correction: A systems view of spliceosomal assembly and branchpoints with iCLIP.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.