Avian influenza A(H5) virus circulation in live bird markets in Vietnam, 2017-2022.

BACKGROUND: Highly pathogenic avian influenza A(H5) human infections are a global concern, with many A(H5) human cases detected in Vietnam, including a case in October 2022. Using avian influenza virus surveillance from March 2017-September 2022, we described the percent of pooled samples that were positive for avian influenza A, A(H5), A(H5N1), A(H5N6), and A(H5N8) viruses in live bird markets (LBMs) in Vietnam. METHODS: Monthly at each LBM, 30 poultry oropharyngeal swab specimens and five environmental samples were collected. Samples were pooled in groups of five and tested for influenza A, A(H5), A(H5N1), A(H5N6), and A(H5N8) viruses by real-time reverse-transcription polymerase chain reaction. Trends in the percent of pooled samples that were positive for avian influenza were summarized by LBM characteristics and time and compared with the number of passively detected avian influenza outbreaks using Spearman's rank correlation. RESULTS: A total of 25,774 pooled samples were collected through active surveillance at 167 LBMs in 24 provinces; 36.9% of pooled samples were positive for influenza A, 3.6% A(H5), 1.9% A(H5N1), 1.1% A(H5N6), and 0.2% A(H5N8). Influenza A(H5) viruses were identified January-December and at least once in 91.7% of sampled provinces. In 246 A(H5) outbreaks in poultry; 20.3% were influenza A(H5N1), 60.2% A(H5N6), and 19.5% A(H5N8); outbreaks did not correlate with active surveillance. CONCLUSIONS: In Vietnam, influenza A(H5) viruses were detected by active surveillance in LBMs year-round and in most provinces sampled. In addition to outbreak reporting, active surveillance for A(H5) viruses in settings with high potential for animal-to-human spillover can provide situational awareness.

Synthesis of (±)-Emtricitabine and (±)-Lamivudine by Chlorotrimethylsilane-Sodium Iodide-Promoted Vorbrüggen Glycosylation.

By simple combination of water and sodium iodide (NaI) with chlorotrimethylsilane (TMSCl), promotion of a Vorbrüggen glycosylation en route to essential HIV drugs emtricitabine (FTC) and lamivudine (3TC) is achieved. TMSCl-NaI in wet solvent (0.1 M water) activates a 1,3-oxathiolanyl acetate donor for N-glycosylation of silylated cytosine derivatives, leading to cis-oxathiolane products with up to 95% yield and >20:1 dr. This telescoped sequence is followed by recrystallization and borohydride reduction, resulting in rapid synthesis of (±)-FTC/3TC from a tartrate diester.

Functional genomics approaches to improve pre-clinical drug screening and biomarker discovery.

Advances in sequencing technology have enabled the genomic and transcriptomic characterization of human malignancies with unprecedented detail. However, this wealth of information has been slow to translate into clinically meaningful outcomes. Different models to study human cancers have been established and extensively characterized. Using these models, functional genomic screens and pre-clinical drug screening platforms have identified genetic dependencies that can be exploited with drug therapy. These genetic dependencies can also be used as biomarkers to predict response to treatment. For many cancers, the identification of such biomarkers remains elusive. In this review, we discuss the development and characterization of models used to study human cancers, RNA interference and CRISPR screens to identify genetic dependencies, large-scale pharmacogenomics studies and drug screening approaches to improve pre-clinical drug screening and biomarker discovery.

Total Synthesis of (±)-Sceptrin.

A four-step synthesis of the dimeric pyrrole-imidazole alkaloid sceptrin is reported. The brevity of the route is based on a simple solution developed for selective assembly of the cyclobutane core of the natural product. The photochemical intermolecular [2 + 2] dimerization of a useful hymenidin surrogate enables direct entry to this enigmatic class of biologically active marine secondary metabolites.

Central nervous system-specific efficacy of CDK4/6 inhibitors in randomized controlled trials for metastatic breast cancer.

Importance: Metastatic breast cancer with central nervous system (CNS) metastases carries a poor prognosis. Recently, CDK4/6 inhibitors have demonstrated a progression free survival (PFS) and overall survival benefit when combined with standard endocrine therapy in advanced hormone receptor (HR)+/HER2- breast cancer. Pre-clinical data suggests possible activity of CDK4/6 inhibitors in the brain, but their CNS-specific benefit has not been explored in clinical practice. Methods: We reviewed clinical trials investigating the efficacy of CDK4/6 inhibitors for advanced or metastatic HR+/HER2- breast cancer. We also reviewed pre-clinical studies that demonstrated the ability of CDK4/6 inhibitors to cross the blood-brain barrier (BBB) and halt the growth of brain metastases in animal models. Findings: An ongoing phase II trial (NCT02308020) was designed to investigate the safety and tolerability of abemaciclib for treatment of patients with CNS metastases, with preliminary data showing partial response in some patients. Review of key randomized phase III trials revealed a scarcity of data pertaining to the development of new CNS metastases. Pre-clinical models demonstrate that CDK4/6 inhibitors are able to cross the BBB and can delay the growth of brain metastases. Conclusions: Despite encouraging pre-clinical evidence, there is a lack of clinical data to inform CNS-specific response rates to CDK4/6 inhibitors among patients with metastatic breast cancer. Given that the treatment of patients with breast cancer brain metastases represents an area of unmet medical need, enrollment of patients with CNS metastases in ongoing clinical trials should be encouraged; innovative trials that examine response of CNS metastases to CDK4/6 inhibitors are also of interest.

Progressive reduction of hospital length of stay following minimally invasive repair of pectus excavatum: A retrospective comparison of three analgesia modalities, the role of addressing patient anxiety, and reframing patient expectations.

PURPOSE: Management of postoperative pain is a significant challenge following the Nuss procedure. Epidurals, PCAs, and newer analgesia modalities have been used elsewhere without demonstrating consistent improvement in the reported length of hospital stays (LOS). We reviewed a large single surgeon experience identifying three different methods of analgesia used over time to highlight marked improvement in patient LOS. METHODS: IRB approval was obtained and patient clinical information was retrospectively reviewed from 2001 to 2017. The primary outcome variable was length of hospital stay. An expanded preoperative consultation reviews the issue of pain, the negative impact of anxiety on recovery, and our current success of shortened hospital stays with our patients. RESULTS: One hundred and seventy-three patients representing three different analgesia approaches had a LOS of 4.4 days (epidural); 2.2 days (PCA/intercostal nerve block); and 1.6 days (scheduled oral pain meds/intercostal nerve blocks). The current LOS for patients is 1.0 day. Patients successfully stop using narcotics by the end of the first week postoperatively. CONCLUSIONS: Intraoperative intercostal nerve blocks, scheduled postoperative pain medications, and enhanced preoperative consultation aimed to educate patients about anxiety and reframe patient pain expectations have collectively decreased LOS, and reduced postoperative narcotic usage. TYPE OF STUDY: Clinical research LEVEL OF EVIDENCE: Level III.

Synthesis of Complex Stereoheptads en Route to Daphnane Diterpene Orthoesters.

Tricyclic cores of the daphnane diterpene orthoesters (DDOs) are synthesized in 10 steps from readily available materials. Key to their assembly is the development of a stereocontrolled p-quinol functionalization sequence which enables rapid access to DDO C-ring stereopolyads from simple precursors. Problems encountered in stereo- and regioselectivity are highlighted and solved by exact changes in choreography, although it is shown that the undesired stereochemical outcomes also proceed with high selectivity.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy.

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Shifting Clade Distribution, Reassortment, and Emergence of New Subtypes of Highly Pathogenic Avian Influenza A(H5) Viruses Collected from Vietnamese Poultry from 2012 to 2015.

Whole-genome sequences of representative highly pathogenic avian influenza A(H5) viruses from Vietnam were generated, comprising samples from poultry outbreaks and active market surveillance collected from January 2012 to August 2015. Six hemagglutinin gene clades were characterized. Clade 1.1.2 was predominant in southern Mekong provinces throughout 2012 and 2013 but gradually disappeared and was not detected after April 2014. Clade 2.3.2.1c viruses spread rapidly during 2012 and were detected in the south and center of the country. A number of clade 1.1.2 and 2.3.2.1c interclade reassortant viruses were detected with different combinations of internal genes derived from 2.3.2.1a and 2.3.2.1b viruses, indicating extensive cocirculation. Although reassortment generated genetic diversity at the genotype level, there was relatively little genetic drift within the individual gene segments, suggesting genetic stasis over recent years. Antigenically, clade 1.1.2, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c viruses remained related to earlier viruses and WHO-recommended prepandemic vaccine strains representing these clades. Clade 7.2 viruses, although detected in only low numbers, were the exception, as indicated by introduction of a genetically and antigenically diverse strain in 2013. Clade 2.3.4.4 viruses (H5N1 and H5N6) were likely introduced in April 2014 and appeared to gain dominance across northern and central regions. Antigenic analyses of clade 2.3.4.4 viruses compared to existing clade 2.3.4 candidate vaccine viruses (CVV) indicated the need for an updated vaccine virus. A/Sichuan/26221/2014 (H5N6) virus was developed, and ferret antisera generated against this virus were demonstrated to inhibit some but not all clade 2.3.4.4 viruses, suggesting consideration of alternative clade 2.3.4.4 CVVs.IMPORTANCE Highly pathogenic avian influenza (HPAI) A(H5) viruses have circulated continuously in Vietnam since 2003, resulting in hundreds of poultry outbreaks and sporadic human infections. Despite a significant reduction in the number of human infections in recent years, poultry outbreaks continue to occur and the virus continues to diversify. Vaccination of poultry has been used as a means to control the spread and impact of the virus, but due to the diversity and changing distribution of antigenically distinct viruses, the utility of vaccines in the face of mismatched circulating strains remains questionable. This study assessed the putative amino acid changes in viruses leading to antigenic variability, underscoring the complexity of vaccine selection for both veterinary and public health purposes. Given the overlapping geographic distributions of multiple, antigenically distinct clades of HPAI A(H5) viruses in Vietnam, the vaccine efficacy of bivalent poultry vaccine formulations should be tested in the future.

Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells.

Most human breast cancers have diversified genomically and biologically by the time they become clinically evident. Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRAS(G12D)), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. DNA barcoding of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop.

DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts.

Genomic and phenotypic analyses indicate extensive intra- as well as intertumoral heterogeneity in primary human malignant cell populations despite their clonal origin. Cellular DNA barcoding offers a powerful and unbiased alternative to track the number and size of multiple subclones within a single human tumour xenograft and their response to continued in vivo passaging. Using this approach we find clone-initiating cell frequencies that vary from ~1/10 to ~1/10,000 cells transplanted for two human breast cancer cell lines and breast cancer xenografts derived from three different patients. For the cell lines, these frequencies are negatively affected in transplants of more than 20,000 cells. Serial transplants reveal five clonal growth patterns (unchanging, expanding, diminishing, fluctuating or of delayed onset), whose predominance is highly variable both between and within original samples. This study thus demonstrates the high growth potential and diverse growth properties of xenografted human breast cancer cells.

Optimized inhibitors of soluble epoxide hydrolase improve in vitro target residence time and in vivo efficacy.

Diabetes is affecting the life of millions of people. A large proportion of diabetic patients suffer from severe complications such as neuropathic pain, and current treatments for these complications have deleterious side effects. Thus, alternate therapeutic strategies are needed. Recently, the elevation of epoxy-fatty acids through inhibition of soluble epoxide hydrolase (sEH) was shown to reduce diabetic neuropathic pain in rodents. In this report, we describe a series of newly synthesized sEH inhibitors with at least 5-fold higher potency and doubled residence time inside both the human and rodent sEH enzyme than previously reported inhibitors. These inhibitors also have better physical properties and optimized pharmacokinetic profiles. The optimized inhibitor selected from this new series displayed improved efficacy of almost 10-fold in relieving pain perception in diabetic neuropathic rats as compared to the approved drug, gabapentin, and previously published sEH inhibitors. Therefore, these new sEH inhibitors could be an attractive alternative to treat diabetic neuropathy in humans.

Integrin ß3 links therapy resistance and cancer stem cell properties.

Heterogeneity in tumour cell properties underlies many treatment failures. Understanding the sources of such heterogeneity has proved to be challenging, but remains critical to improving patient outcomes. Integrin α(v)ß3 expression in multiple types of solid tumour stem cells is now shown to control a pro-survival pathway that contributes to therapy resistance.

Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets.

Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.

Clonal analysis via barcoding reveals diverse growth and differentiation of transplanted mouse and human mammary stem cells.

Cellular barcoding offers a powerful approach to characterize the growth and differentiation activity of large numbers of cotransplanted stem cells. Here, we describe a lentiviral genomic-barcoding and analysis strategy and its use to compare the clonal outputs of transplants of purified mouse and human basal mammary epithelial cells. We found that both sources of transplanted cells produced many bilineage mammary epithelial clones in primary recipients, although primary clones containing only one detectable mammary lineage were also common. Interestingly, regardless of the species of origin, many clones evident in secondary recipients were not detected in the primary hosts, and others that were changed from appearing luminal-restricted to appearing bilineage. This barcoding methodology has thus revealed conservation between mice and humans of a previously unknown diversity in the growth and differentiation activities of their basal mammary epithelial cells stimulated to grow in transplanted hosts.

Descriptive results of a prospective cohort study of avian influenza in the Mekong River Delta of Viet Nam.

A prospective cohort study of avian influenza infection in poultry flocks was carried out in the Mekong River Delta of Viet Nam between December 2008 and April 2010. Our objectives were to (i) estimate the prevalence and incidence of avian influenza virus infection and (ii) assess the efficacy of H5N1 vaccination programmes as indicated by the presence of H5 antibody in vaccinated and unvaccinated poultry. Real-time PCR and H5 multiplex assays were used to detect the antigen of avian influenza viruses from swab samples. The haemagglutination inhibition test was used to detect H5 antibody. A total of 17 968 swab and 14 878 blood samples were collected from 5476 birds over the study period. The overall incidence rate of influenza type A virus infection was 5 (95% CI 4-7) positive birds per 100 bird-months at risk. The overall incidence rate of H5 virus infection was 0.2 (95% CI 0.1-0.5) positive birds per 100 bird-months at risk. Fifty (95% CI 48-52) birds per 100 tested birds were H5 HI positive in the unvaccinated group compared with 71 (95% CI 69-73) birds per 100 in the vaccinated group. Influenza type A and H5 viruses were circulating in village poultry throughout the study period with no recorded signs of clinical disease. This implies that interventions need to be carried out continuously throughout the year rather than only focusing on the established high-risk periods. Broiler ducks had an incidence rate of influenza H5 virus infection approximately four times greater than that of layer ducks and in-contact species. We conclude that broiler ducks are likely to be the main entry route for H5 virus into poultry flocks in the MRD. Control efforts would benefit from understanding why there is a difference between villages in H5 incidence and developing strategies to provide greater protection to broiler ducks.

Prevalence and distribution of avian influenza a(H5N1) virus clade variants in live bird markets of Vietnam, 2011-2013.

Active surveillance for avian influenza (Al) viruses in poultry sold at live bird markets (LBMs) was conducted in 44 of 63 provinces throughout Vietnam over two periods from September 2011 to February 2012 and October 2012 to June 2013. The study objectives were to assess the prevalence of avian influenza type A, H5, and H5N1 subtype viruses and characterize the geographical and temporal distribution of H5N1 virus genetic variants across the country. Monthly sampling was conducted in 394 LBMs located in 372 communes. A total of 9790 oropharyngeal swabs from poultry were screened for influenza A virus by real-time reverse-transcriptase PCR Virus isolation was attempted on all positive samples in embryonated chicken eggs, and the HA1 region of each H5 virus isolate was sequenced. Market prevalence of H5 subtype virus was 32.2% (127/394) over the cumulative 15 mo of surveillance. Phylogenetic analyses indicated that clade 1.1 viruses persisted in the south, whereas three genetically distinct subgroups of dade 2.3.2.1 were found simultaneously in northern, central, and southern Vietnam. Clade 2.3.2.1c viruses first appeared in July 2012 and spread rapidly to the center and south of Vietnam in late 2012, where they were predominant among clade 2.3.2.1 viruses and were detected in both active LBM surveillance and poultry outbreaks. Given the overlapping geographic distribution of dade variants and the antigenic divergence previously described for these dades, current AI poultry vaccines used in Vietnam may require bivalent formulations containing representatives of both dade 1.1 and dade 2.3.2.1 viruses.