Shigella sonnei utilises colicins during inter-bacterial competition.

The mammalian colon is one of the most densely populated habitats currently recognised, with 1011-1013 commensal bacteria per gram of colonic contents. Enteric pathogens must compete with the resident intestinal microbiota to cause infection. Among these enteric pathogens are Shigella species which cause approximately 125 million infections annually, of which over 90 % are caused by Shigella flexneri and Shigella sonnei. Shigella sonnei was previously reported to use a Type VI Secretion System (T6SS) to outcompete E. coli and S. flexneri in in vitro and in vivo experiments. S. sonnei strains have also been reported to harbour colicinogenic plasmids, which are an alternative anti-bacterial mechanism that could provide a competitive advantage against the intestinal microbiota. We sought to determine the contribution of both T6SS and colicins to the anti-bacterial killing activity of S. sonnei. We reveal that whilst the T6SS operon is present in S. sonnei, there is evidence of functional degradation of the system through SNPs, indels and IS within key components of the system. We created strains with synthetically inducible T6SS operons but were still unable to demonstrate anti-bacterial activity of the T6SS. We demonstrate that the anti-bacterial activity observed in our in vitro assays was due to colicin activity. We show that S. sonnei no longer displayed anti-bacterial activity against bacteria that were resistant to colicins, and removal of the colicin plasmid from S. sonnei abrogated anti-bacterial activity of S. sonnei. We propose that the anti-bacterial activity demonstrated by colicins may be sufficient for niche competition by S. sonnei within the gastrointestinal environment.

Expression of NEDD4L and ENaC in Urinary Extracellular Vesicles in Pre-eclampsia.

OBJECTIVE: To assess changes in expression of renal epithelial sodium channel (ENaC) and NEDD4L, a ubiquitin ligase, in urinary extracellular vesicles (UEV) of pre-eclamptic women compared to normal pregnant controls. METHODS: Urine was collected from pre-eclamptic women (PE, n = 20) or during normal pregnancy (NP, n = 20). UEV were separated by differential ultracentrifugation. NEDD4L, α-ENaC and γ-ENaC were identified by immunoblotting. RESULTS: There was no difference in the expression of NEDD4L (p = 0.17) and α-ENaC (p = 0.10). PE subjects showed increased expression of γ-ENaC by 6.9-fold compared to NP (p < 0.0001). CONCLUSION: ENaC expression is upregulated in UEV of pre-eclamptic subjects but was not associated with changes in NEDD4L.

Anti-methicillin resistance Staphylococcus aureus and in vitro toxicology evaluation of corilagin-loaded gelatin/agar microspheres with potential biotextile applications.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged since the early 1960s. The increasing resistance of pathogens to currently used antibiotics requires the urgent discovery of new antimicrobials effective in combating drug-resistant bacteria. From past to present, medicinal plants are useful to cure human diseases. Corilagin (ß-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-d-glucose), commonly found in Phyllanthus species, exerts potentiating effect on ß-lactams against MRSA. However, its biological effect may not be fully utilized. Therefore, incorporating microencapsulation technology with the delivery of corilagin would be more effective in utilizing the potential effect on biomedical applications. This work reports the development of a safe micro-particulate system which combined agar with gelatin as wall matrix materials for topical delivery of corilagin in order to eliminate the potential toxicity of the crosslinker formaldehyde. The optimal parameters for microsphere preparation were identified and the particle size of optimal microspheres was 20.11 µm ± 3.58. Antibacterial studies revealed that micro-trapped corilagin (minimum bactericidal concentration, MBC = 0.5 mg/mL) possessed a higher potency against MRSA than free corilagin (MBC = 1 mg/mL). The in vitro skin cytotoxicity showed the safety of the corilagin-loaded microspheres for topical applications, with approximately 90 % of HaCaT cell viability. Our results demonstrated the potential of corilagin-loaded gelatin/agar microspheres for the applicable bio-textile products to treat drug-resistant bacterial infections.

Robust deep learning-based protein sequence design using ProteinMPNN.

Although deep learning has revolutionized protein structure prediction, almost all experimentally characterized de novo protein designs have been generated using physically based approaches such as Rosetta. Here, we describe a deep learning-based protein sequence design method, ProteinMPNN, that has outstanding performance in both in silico and experimental tests. On native protein backbones, ProteinMPNN has a sequence recovery of 52.4% compared with 32.9% for Rosetta. The amino acid sequence at different positions can be coupled between single or multiple chains, enabling application to a wide range of current protein design challenges. We demonstrate the broad utility and high accuracy of ProteinMPNN using x-ray crystallography, cryo-electron microscopy, and functional studies by rescuing previously failed designs, which were made using Rosetta or AlphaFold, of protein monomers, cyclic homo-oligomers, tetrahedral nanoparticles, and target-binding proteins.

Nano-Graphene Layer from Facile, Scalable and Eco-Friendly Liquid Phase Exfoliation Strategy as Effective Barrier Layer for High-Performance and Durable Direct Liquid Alcohol Fuel Cells.

Graphene, in spite of exceptional physio-chemical properties, still faces great limitations in its use and industrial scale-up as highly selective membranes (enhanced ratio of proton conductivity to fuel cross-over) in liquid alcohol fuel cells (LAFCs), due to complexity and high cost of prevailing production methods. To resolve these issues, a facile, low-cost and eco-friendly approach of liquid phase exfoliation (bath sonication) of graphite to obtain graphene and spray depositing the prepared graphene flakes, above anode catalyst layer (near the membrane in the membrane electrode assembly (MEA)) as barrier layer at different weight percentages relative to the base membrane Nafion 115 was utilized in this work. The 5 wt.% nano-graphene layer raises 1 M methanol/oxygen fuel cell power density by 38% to 91 mW·cm-2, compared to standard membrane electrode assembly (MEA) performance of 63 mW·cm-2, owing to less methanol crossover with mild decrease in proton conductivity, showing negligible voltage decays over 20 h of operation at 50 mA·cm-2. Overall, this work opens three prominent favorable prospects: exploring the usage of nano-materials prepared by liquid phase exfoliation approach, their effective usage in ion-transport membrane region of MEA and enhancing fuel cell power performance.

Prevalences of levofloxacin resistance and pncA mutation in isoniazid-resistant Mycobacterium tuberculosis in Hong Kong.

INTRODUCTION: In 2018, the World Health Organization recommended a 6-month treatment regimen that included levofloxacin and pyrazinamide for isoniazid-resistant Mycobacterium tuberculosis without rifampicin resistance (Hr-TB). Susceptibility testing for both drugs is not routinely performed for Hr-TB in Hong Kong. This study examined the prevalences of levofloxacin and pyrazinamide resistances in Hr-TB and explored associated risk factors. METHODS: All Hr-TB isolates archived during 2018 were retrieved. Isolates were de-duplicated to identify unique cases. Levofloxacin susceptibility testing was performed using the MGIT 960 System; pncA gene sequencing was used as a surrogate indicator of pyrazinamide susceptibility. Previous laboratory records for each case were analysed. RESULTS: In total, 160 phenotypic Hr-TB cases were identified from among 3411 patients with tuberculosis (4.7%). Among these, 157 were analysed, revealing 0.6% (n=1) levofloxacin resistance and 4.5% (n=7) pyrazinamide resistance, respectively. Independent risk factors associated with pncA mutations included history of tuberculosis in the affected patient and isoniazid poly-resistance (ie, double and triple resistances), but not mono-resistance. CONCLUSION: For Hr-TB in Hong Kong, levofloxacin resistance is rare and pyrazinamide resistance-associated pncA mutations are uncommon. Routine susceptibility testing for these drugs is not indicated unless related risk factors are identified.

Exercise is Medicine® Canada on Campus Casebook: Evaluation of a novel knowledge translation tool and lessons learned.

The Exercise is Medicine® Canada on Campus (EIMC-OC) program aims to integrate exercise prescription into healthcare and encourage students to implement physical activity initiatives on campus. However, multi-site interventions like EIMC-OC are often challenged with communicating and sharing strategies across geographically dispersed groups. The EIMC-OC Casebook was created as an accessible method to enhance program success by sharing ideas and implementation strategies between groups, but its potential utility is unknown as few studies have evaluated casebooks. This study evaluated the usability and value of the EIMC-OC Casebook for promoting physical activity and established end-users' insight on Casebook future directions. The Casebook was shared and semi-structured interviews were conducted with established and developing EIMC-OC groups. Five themes discussing the usability, value, and future directions of the Casebook were identified. Participants implemented the Casebook to varying degrees, found it to be a valuable communication medium, and recommended revisions, which may enhance its implementation. The EIMC-OC Casebook is a valuable tool that exemplifies campus-based efforts to promote physical activity, augments between-group communication, and helps groups conduct effective initiatives. Program leaders and researchers may benefit from a similar Casebook approach, and recommendations are provided to evaluators aiming to enhance the effectiveness of multi-site programs.

Molecular detection of Mycoplasma genitalium in endocervical swabs and associated rates of macrolide and fluoroquinolone resistance in Hong Kong.

INTRODUCTION: There is a global trend of increasing macrolide and fluoroquinolone resistance in Mycoplasma genitalium (MG), such that international guidelines recommend molecular detection of resistance if a patient has MG-positive test results. Tests for MG are not routinely performed in Hong Kong. This study examined the detection of MG in endocervical swabs and the associated macrolide and fluoroquinolone resistance rates. METHODS: Endocervical swabs received from two sexual health clinics in Hong Kong for routine assessments of Chlamydia trachomatis and Neisseria gonorrhoeae were also subjected to detection of MG. All MG-positive samples were tested for resistance-mediating mutations in 23S rRNA, parC, and gyrA genes. Laboratory records and past results for each patient were analysed. RESULTS: In total, endocervical swabs from 285 patients were included in this study. Mycoplasma genitalium was detected in swabs from 21 patients (7.4%) by real-time polymerase chain reaction with a commercial kit. Among MG-positive samples which were successfully analysed further, macrolide resistance-mediating mutations in 23S rRNA were found in 42.1% (8/19); fluoroquinolone resistance-related mutations in parC and gyrA were found in 65% (13/20) and 0% (0/20), respectively. All macrolide-resistant MG strains were also fluoroquinolone-resistant (42.1%, 8/19). No assessed factors were associated with the detection of MG or resistance-related mutations. CONCLUSION: In Hong Kong, MG was detected in endocervical swabs from 7.4% of patients in sexual health clinics, with high rates of macrolide and fluoroquinolone resistance. These findings warrant careful review of testing, clinical correlation, and treatment strategies for MG in the context of increasing antibiotic resistance.

Swine ANP32A Supports Avian Influenza Virus Polymerase.

Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as "mixing vessels," being susceptible to both avian- and human-origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study, we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports the activity of avian-origin influenza virus polymerases and avian influenza virus replication. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic, it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the enhanced proviral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the leucine-rich repeat and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the mixing vessel trait of swine and further our understanding of the evolution and ecology of viruses in this host.IMPORTANCE Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but they only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus "mixing vessels." In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this by binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as mixing vessels.

Elderly males with or without existing osteoporotic vertebral fracture have much lower future vertebral fracture risk than elderly females: the MrOS (Hong Kong) year-4 follow-up spine radiograph study.

MrOS (Hong Kong)'s year-4 follow-up shows, for subjects at baseline without vertebral deformity (VD) and endplate or/and cortex fracture (ECF), the VD progression/new VD rate during follow-up in males was half of our paired MsOS (Hong Kong) study's results. For those with VD or ECF, the VD progression/new VD was less than one sixth of females' rate. INTRODUCTION: This study documents MrOS (Hong Kong)'s year-4 follow-up, and the results are compared with the MsOS (Hong Kong) study. Of elderly females with Genant's grade-0, -1, -2, and -3 VD, at year-4 follow-up, 4.6%, 8%, 10.6%, and 28.9% had at least one VD progression or incident VD, respectively. METHODS: Spine radiographs of 1500 Chinese males with baseline (mean age 71.7 years, range 65-91 years) and year-4 follow-up were evaluated according to Genant's VD criteria and ECF (non-existent, ECF0; or existent, ECF1). Grade-2 VDs were divided into mild (VD2m, 25-34% height loss) and severe (VD2s, 34-40% height loss) subgroups. Study subjects were graded into eight categories: VD0/ECF0, VD1/ECF0, VD2m/ECF0, VD0/ECF1, VD1/ECF1, VD2m/ECF1, VD2s/ECF1, and VD3/ECF1. With an existing VD, a further height loss of ≥ 15% was a VD progression. A new VD incident was a change from grade-0 to grade-2/3, or to grade-1 with ≥ 10% height loss. RESULTS: Of subjects with Genant's grade-0, 2.05% (25/1219) developed at least one VD progression or/and new VD, while of subjects with Genant's grade-1, -2, and -3 VD, only 2% (3/149), 3.1% (3/96), and 2.8% (1/36) developed at least one VD progression/new VD, respectively. Among the three ECF0 groups, there was a significant difference in new ECF incidence, with VD0/ECF0 being the lowest and VD2m/ECF0 being the highest. CONCLUSION: VD progression/new VD is much less common in elderly men than in elderly women. Vertebrae with VD had a higher risk of developing ECF.

Kisspeptin regulation of human decidual stromal cells motility via FAK-Src intracellular tyrosine kinases.

STUDY QUESTION: Can of Clinical Genetics, Maastricht University Medical Centre, Maastricht kisspeptin and its analogues regulate the motility of human decidual stromal cells and what intracellular signaling pathways are involved? SUMMARY ANSWER: Kisspeptin analogue-mediated cell motility in human decidual stromal cells via the focal adhesion kinase (FAK)-steroid receptor coactivator (Src) pathway suggesting that kisspeptin may modulate embryo implantation and decidual programming in human pregnancy. WHAT IS KNOWN ALREADY: The extravillous trophoblast invades the maternal decidua during embryo implantation and placentation. The motile behavior and invasive potential of decidual stromal cells regulate embryo implantation and programming of human pregnancy. STUDY DESIGN, SIZE, DURATION: Human decidual stromal cells were isolated from healthy women undergoing elective termination of a normal pregnancy at 6- to 12-week gestation, after informed consent. PARTICIPANTS/MATERIALS, SETTING, METHODS: Kisspeptin analogues were synthetic peptides. Cell motility was estimated by an invasion and migration assay. Immunoblot analysis was performed to investigate the expression of kisspeptin receptor and the effects of kisspeptin analogues on the phosphorylation of FAK and Src. Small interfering RNAs (siRNAs) were used to knock down the expression of kisspeptin receptor, FAK, Src, matrix metallo-proteinases (MMPs) 2 and 9, and extracellular signal-regulated protein kinase (ERK) 1/2. MAIN RESULTS AND THE ROLE OF CHANCE: The kisspeptin receptor was expressed in human decidual stromal cells. Kisspeptin agonist decreased, but antagonist increased, cell motility. Kisspeptin agonist decreased the phosphorylation of FAK and Src tyrosine kinases, whereas antagonist increased it. These effects on phosphorylation were abolished by kisspeptin receptor siRNA. The activation of cell motility by kisspeptin analogues was suppressed by siRNA knockdown of endogenous FAK (decreased 66%), Src (decreased 60%), kisspeptin receptor (decreased 26%), MMP-2 (decreased 36%), MMP-9 (decreased 23%), and ERK 1/2 inhibitor (decreased 27%). LIMITATIONS, REASONS FOR CAUTION: Human decidual stromal cells were obtained from women having terminations after 6-12 weeks of pregnancy and differences in timing could affect their properties. WIDER IMPLICATIONS OF THE FINDINGS: Kisspeptin acting within the endometrium has a potential modulatory role on embryo implantation and decidual programming of human pregnancy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grant NSC-104-2314-B-182A-146-MY2 (to H.-M.W.) from the Ministry of Science and Technology, Taiwan, and grants CMRPG3E0401 and CMRPG3E0402 (to H.-M.W.). This work was also supported by grants from the Canadian Institutes of Health Research to P.C.K.L. P.C.K.L. is the recipient of a Child & Family Research Institute Distinguished Investigator Award. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Uncertainty Quantification for Flow and Transport in Highly Heterogeneous Porous Media Based on Simultaneous Stochastic Model Dimensionality Reduction.

Groundwater flow models are usually subject to uncertainty as a consequence of the random representation of the conductivity field. In this paper, we use a Gaussian process model based on the simultaneous dimension reduction in the conductivity input and flow field output spaces in order quantify the uncertainty in a model describing the flow of an incompressible liquid in a random heterogeneous porous medium. We show how to significantly reduce the dimensionality of the high-dimensional input and output spaces while retaining the qualitative features of the original model, and secondly how to build a surrogate model for solving the reduced-order stochastic model. A Monte Carlo uncertainty analysis on the full-order model is used for validation of the surrogate model.

Osteoporotic vertebral deformity with endplate/cortex fracture is associated with higher further vertebral fracture risk: the Ms. OS (Hong Kong) study results.

Compared with vertebrae without deformity, vertebrae with mild/moderate deformity have a higher risk of endplate or/and cortex fracture (ecf). Compared with subjects without ecf, subjects with ecf are at a higher risk of short-term (4-year period) deformity progression and new incident deformity. INTRODUCTION: The progression and incidence of osteoporotic vertebral deformity/fracture (VD/VF) in elderly Chinese females remain not well documented. METHODS: Spine radiographs of 1533 Chinese females with baseline and year-4 follow-up (mean age 75.7 years) were evaluated according to Genant's VD criteria and endplate/cortex fracture (non-existent: ecf0 or existent: ecf1). Grade-2 VDs were divided into mild (vd2m, 25-34% height loss) and severe (vd2s, 34-40% height loss) subgroups. According to their VD/VF, subjects were graded into seven categories: vd0/ecf0, vd1/ecf0, vd2m/ecf0, vd1/ecf1, vd2m/ecf1, vd2s/ecf1, and vd3/ecf1. With an existing VD, a further height loss of ≥ 15% was a VD progression. A new incident VD was a change from grade-0 to grade-2/3 or to grade-1 with ≥ 10% height loss. RESULTS: Of subjects with Genant's grades 0, - 1, - 2, and - 3 VD, at follow-up, 4.6%, 8%, 10.6%, and 28.9% had at least one VD progression or new incident VD respectively. Among the three ecf0 groups, there was no difference in VD progression or new VD; while there was a significant difference in new ecf incidence, with vd0/ecf0 being lowest and vd2m/ecf0 being highest. Vd1/ecf0 and vd2m/ecf0 vertebrae had a higher risk of turning to ecf1 than vd0/ecf0 vertebrae. If vd1/ecf0 and vd2m/ecf0 subjects were combined together (range 20-34% height loss) to compare with vd1/ecf1 and vd2m/ecf1 subjects, the latter had significantly higher VD progression and new VD rates. CONCLUSION: Vertebrae with grade-1/2 VDs had a higher risk of developing ECF. Subjects with pre-existing ECFs had a higher risk of worsening or new vertebral deformities.

Pathogen infections and primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a multi-factorial disease caused by the interaction of both genetic predisposition and environmental triggers. Bacterial infection has been investigated most intensively, both epidemiologically and experimentally, as a prime environmental aetiology in PBC. The association of recurrent history of urinary tract infection (UTI) with PBC has been frequently confirmed by several large-scale, case-control studies, despite variation in geographic area or case-finding methods. Escherichia coli is a predominant pathogen in most cases with UTI. Animal studies and molecular mimicry analysis between the human and E. coli E2 subunit of the 2-oxo-acid dehydrogenase complexes demonstrated that E. coli infection is a key factor in breaking immunological tolerance against the mitochondria, resulting in the production of anti-mitochondrial autoantibodies (AMA), the disease-specific autoantibodies of PBC. Novosphingobium aromaticivorans, a ubiquitous xenobiotic-metabolizing bacterium, is another candidate which may be involved in the aetiology of PBC. Meanwhile, improved environmental hygiene and increased prevalence of PBC, especially in males, may argue against the aetiological role of bacterial infection in PBC. Multiple mechanisms can result in the loss of tolerance to mitochondrial autoantigens in PBC; nonetheless, bacterial infection is probably one of the dominant pathways, especially in female patients. Notably, there is a rising prevalence of male patients with PBC. With increasing exposure to environmental xenobiotics in both genders, studies directed towards identifying the environmental culprit with systematically designed case-control studies are much needed to further determine the environmental factors and role of bacterial infections in PBC.

No Effects of Black Tea on Cognitive Decline Among Older US Men: A Prospective Cohort Study.

BACKGROUND: Accumulating evidence supports the neuroprotective effects of bioactive compounds from tea leaves. There are limited data from black tea consumption populations. OBJECTIVE: To determine whether black tea consumption is associated with cognitive decline among older men. METHODS: We chose to study the association between black tea consumption and cognition using data from the Osteoporotic Fractures in Men (MrOS) cohort, which collected information on tea consumption at baseline and has repeatedly assessed cognitive function in 3,844 men aged 65+ years (mean = 72.4 years). We defined tea drinkers as those who drank black tea at least once per week and further grouped them into weekly drinkers and daily drinkers. Cognitive function was assessed at baseline and approximately 7 years later using the Modified Mini-Mental State Examination (3MSE). Multivariable logistic regression and linear regression models were constructed to assess the association between black tea consumption and risk of fast cognitive decline as a binary variable and change in 3MSE scores as continuous variable. Fast cognitive decline was defined as decline in 3MSE >1.5 standard deviation of mean change score. Models were adjusted for age, education level, and baseline cognitive scores. RESULTS: Weekly and daily black tea drinkers were 24.8% and 12.4% of the study cohort, respectively. Fast cognitive decline occurred in 243 (6.3%) participants. Tea consumption was not associated with risk of cognitive decline, nor was tea associated with cognitive decline measured by absolute change in 3MSE scores. CONCLUSIONS: There was no association of black tea consumption and cognitive decline among older men in the US.

Open versus laparoscopic appendicectomy in acute appendicitis: results of a district general hospital.

BACKGROUND: Despite multiple studies comparing the two methods, the real advantages of laparoscopic appendicectomy (LA) compared to open appendicectomy (OA) are still unclear. Purpose of the current study was to compare the results between the two techniques in a district general hospital. METHODS: The electronic records of all patients who underwent OA or LA in a one year period were reviewed retrospectively. The comparative data points assessed included age, gender, overall complications, length of stay and Clavien-Dindo Classification of Surgical Complications, including the rates of the main types of complications. RESULTS: 300 patients were included in the study. 166 patients underwent OA and 134 patients LA. Postoperative complications were documented in 26 patients (8.7%). LA was employed predominantly in female patients (p = 0.004) and in older patients (p = 0.0015) and was associated with significantly more negative appendicectomies than OA (p = 0.002). No statistically significant difference was noted with regards to the length of hospital stay (p = 0.577), overall postoperative morbidity (p = 0.543) and grading of complications (p = 0.460). Finally, following comparison of the incidence of specific types of complications, only wound infections were significantly different, in favour of LA. CONCLUSION: LA is safe and effective, however, besides the lower incidence of wound sepsis, demonstrates no clear advantage over OA. The selection between OA and LA should thus be tailored to the clinical scenario and the surgeon's preference.

Isodon eriocalyx and its bioactive component Eriocalyxin B enhance cytotoxic and apoptotic effects of gemcitabine in pancreatic cancer.

BACKGROUND: Pancreatic cancer, associated with poor prognosis and low survival rate, has been the fourth leading cause of cancer-related death in the US. Although gemcitabine (Gem) is the first-line chemotherapeutic drug in the management of pancreatic cancer, the median survival extension is only 1.5 months, indicating unsatisfactory clinical results. Therefore, exploring agents that can enhance the anti-cancer activity of Gem would be an attractive strategy. PURPOSE: Our previous studies have demonstrated that eriocalyxin b (EriB), an ent­kaurane diterpenoid isolated from Isodon eriocalyx (Dunn.) Hara, possesses anti-pancreatic cancer effects, thus acting as a potential therapeutic agent. In this study, we further investigated whether EriB or the ethanol extract of I. eriocalyx (Isodon) could potentiate the cytotoxic activity of Gem in human pancreatic adenocarcinoma cells. In addition, the mechanism associated with their effects was also studied. METHODS: The anti-proliferation effect was assessed by MTT assay and Ki-67 immunostaining. The combination effect (addition, synergism and antagonism) of various agents was calculated by the Calcusyn software (Biosoft), utilizing the T.C. Chou Method. Apoptosis was detected using Annexin V and PI double staining followed by quantitative flow cytometry. Protein expression regulated by various treatments was analyzed by western blotting. RESULTS: The combination index revealed that Gem and EriB (or Isodon extract) had synergistic anti-proliferative effect. Both cellular apoptotic and anti-proliferative effects of Gem were significantly increased after combination with EriB (or Isodon extract). The underlying mechanisms involved in the combination effects were elucidated, which include: (1) increased activation of the caspase cascade; (2) reduction of PDK1 and AKT phosphorylation; (3) induction of JNK phosphorylation by Isodon and Gem combination. CONCLUSION: Gem and EriB (or Isodon extract) taken together in combination regulated PDK1/AKT1/caspase and JNK signaling and promoted apoptosis synergistically, which may contribute to the much increased anti-proliferative activity compared to either agent alone.