Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM

Large-scale expansion of highly functional adult human mesenchymal stem cells (aMSCs) remains technologically challenging as aMSCs lose self renewal capacity and multipotency during traditional long-term culture and their quality/quantity declines with donor age and disease. Identification of culture conditions enabling prolonged expansion and rejuvenation would have dramatic impact in regenerative medicine. aMSC-derived decellularized extracellular matrix (ECM) has been shown to provide such microenvironment which promotes MSC self renewal and “stemness”. Since previous studies have demonstrated superior proliferation and osteogenic potential of human fetal MSCs (fMSCs), we hypothesize that their ECM may promote expansion of clinically relevant aMSCs. We demonstrated that aMSCs were more proliferative (∼1.6×) on fMSC-derived ECM than aMSC-derived ECMs and traditional tissue culture wares (TCPS). These aMSCs were smaller and more uniform in size (median ± interquartile range: 15.5 ± 4.1 μm versus 17.2 ± 5.0 μm and 15.5 ± 4.1 μm for aMSC ECM and TCPS respectively), exhibited the necessary biomarker signatures, and stained positive for osteogenic, adipogenic and chondrogenic expressions; indications that they maintained multipotency during culture. Furthermore, fMSC ECM improved the proliferation (∼2.2×), size (19.6 ± 11.9 μm vs 30.2 ± 14.5 μm) and differentiation potential in late-passaged aMSCs compared to TCPS. In conclusion, we have established fMSC ECM as a promising cell culture platform for ex vivo expansion of aMSCs.Singapore-MIT Alliance for Research and Technolog

A one health approach to strengthening antimicrobial stewardship in Wakiso District, Uganda

Antimicrobial stewardship (AMS), as one of the global strategies to promote responsible use of antimicrobials to prevent antimicrobial resistance (AMR), remains poor in many low-and middle-income countries (LMICs). We implemented a project aimed at strengthening AMS in Wakiso district, Uganda using a One Health approach. A total of 86 health practitioners (HPs), including animal health workers, and 227 community health workers (CHWs) participated in training workshops, and over 300 pupils from primary schools were sensitized on AMR, AMS, and infection prevention and control (IPC). We further established two multidisciplinary online communities of practice (CoPs) for health professionals and students, with a current membership of 321 and 162, respectively. In addition, a Medicine and Therapeutics Committee (MTC) was set up at Entebbe Regional Referral Hospital. The project evaluation, conducted three months after training, revealed that the majority of the HPs (92.2%) and CHWs (90.3%) reported enhanced practices, including improved hand washing (57.3% and 81.0%, respectively). In addition, 51.5% of the HPs reported a reduction in the quantity of unnecessary antibiotics given per patient. This project demonstrates that AMS interventions using a One Health approach can promote understanding of the prudent use of antimicrobials and improve practices at health facilities and in communities

Bottom mixed layer oxygen dynamics in the Celtic Sea

The seasonally stratified continental shelf seas are highly productive, economically important environments which are under considerable pressure from human activity. Global dissolved oxygen concentrations have shown rapid reductions in response to anthropogenic forcing since at least the middle of the twentieth century. Oxygen consumption is at the same time linked to the cycling of atmospheric carbon, with oxygen being a proxy for carbon remineralisation and the release of CO2. In the seasonally stratified seas the bottom mixed layer (BML) is partially isolated from the atmosphere and is thus controlled by interplay between oxygen consumption processes, vertical and horizontal advection. Oxygen consumption rates can be both spatially and temporally dynamic, but these dynamics are often missed with incubation based techniques. Here we adopt a Bayesian approach to determining total BML oxygen consumption rates from a high resolution oxygen time-series. This incorporates both our knowledge and our uncertainty of the various processes which control the oxygen inventory. Total BML rates integrate both processes in the water column and at the sediment interface. These observations span the stratified period of the Celtic Sea and across both sandy and muddy sediment types. We show how horizontal advection, tidal forcing and vertical mixing together control the bottom mixed layer oxygen concentrations at various times over the stratified period. Our muddy-sand site shows cyclic spring-neap mediated changes in oxygen consumption driven by the frequent resuspension or ventilation of the seabed. We see evidence for prolonged periods of increased vertical mixing which provide the ventilation necessary to support the high rates of consumption observed

The Use of Orthologous Sequences to Predict the Impact of Amino Acid Substitutions on Protein Function

Computational predictions of the functional impact of genetic variation play a critical role in human genetics research. For nonsynonymous coding variants, most prediction algorithms make use of patterns of amino acid substitutions observed among homologous proteins at a given site. In particular, substitutions observed in orthologous proteins from other species are often assumed to be tolerated in the human protein as well. We examined this assumption by evaluating a panel of nonsynonymous mutants of a prototypical human enzyme, methylenetetrahydrofolate reductase (MTHFR), in a yeast cell-based functional assay. As expected, substitutions in human MTHFR at sites that are well-conserved across distant orthologs result in an impaired enzyme, while substitutions present in recently diverged sequences (including a 9-site mutant that “resurrects” the human-macaque ancestor) result in a functional enzyme. We also interrogated 30 sites with varying degrees of conservation by creating substitutions in the human enzyme that are accepted in at least one ortholog of MTHFR. Quite surprisingly, most of these substitutions were deleterious to the human enzyme. The results suggest that selective constraints vary between phylogenetic lineages such that inclusion of distant orthologs to infer selective pressures on the human enzyme may be misleading. We propose that homologous proteins are best used to reconstruct ancestral sequences and infer amino acid conservation among only direct lineal ancestors of a particular protein. We show that such an “ancestral site preservation” measure outperforms other prediction methods, not only in our selected set for MTHFR, but also in an exhaustive set of E. coli LacI mutants

Quantitative Analysis and Diagnostic Significance of Methylated SLC19A3 DNA in the Plasma of Breast and Gastric Cancer Patients

Background: Previously, we have examined the methylation status of SLC19A3 (solute carrier family 19, member 3) promoter and found that SLC19A3 was epigenetically down-regulated in gastric cancer. Here, we aim to develop a new biomarker for cancer diagnosis using methylated SLC19A3 DNA in plasma. Methodology/Principal Findings: SLC19A3 gene expression was examined by RT-qPCR. Methylation status of SLC19A3 promoter was evaluated by methylation-specific qPCR. SLC19A3 expression was significantly down-regulated in 80% (12/15) of breast tumors (P<0.005). Breast tumors had significant increase in methylation percentage when compared to adjacent non-tumor tissues (P<0.005). A robust and simple methylation-sensitive restriction enzyme digestion and real-time quantitative PCR (MSRED-qPCR) was developed to quantify SLC19A3 DNA methylation in plasma. We validated this biomarker in an independent validation cohort of 165 case-control plasma including 60 breast cancer, 45 gastric cancer patients and 60 healthy subjects. Plasma SLC19A3 methylated DNA level was effective in differentiating both breast and gastric cancer from healthy subjects. We further validated this biomarker in another independent blinded cohort of 78 plasma including 38 breast cancer, 20 gastric cancer patients and 20 healthy subjects. The positive predictive values for breast and gastric cancer were 90% and 85%, respectively. The negative predictive value of this biomarker was 85%. Elevated level in plasma has been detected not only in advanced stages but also early stages of tumors. The positive predictive value for ductal carcinoma in situ (DCIS) cases was 100%. Conclusions: These results suggested that aberrant SLC19A3 promoter hypermethylation in plasma may be a novel biomarker for breast and gastric cancer diagnosis. © 2011 Ng et al.published_or_final_versio

Knowledge, perceptions and practices on antimicrobial resistance in humans and animals in Wakiso district, Uganda: a cross sectional study

Despite increasing evidence on antimicrobial resistance (AMR), there is limited literature on antimicrobial access and use in humans and animals in community settings globally. This study assessed knowledge and perceptions of AMR, as well as practices relating to the use of antimicrobials in humans and animals in Wakiso district, Uganda. This was a cross-sectional study among 418 participants that employed quantitative data collection methods. A structured questionnaire that included questions on knowledge, perceptions, practices related to AMR, and perceptions on access to antimicrobials in humans and animals was used. Data was analysed in STATA version 10. The majority of participants 63.6% (266/418) had heard about AMR mainly from family and friends 57.5% (153/266), and most 70.8% (296/418) were aware that resistant microorganisms cause infections that are difficult to treat. Most participants 62.7% (262/418) thought that they should complete the full dose of antimicrobials when on treatment. However, on the last occasion of antimicrobial use, 13.0% (44/338) revealed that they did not complete the full course of treatment. Participants who were single (APR = 1.12, C.I = 1.03–1.12, p-value = 0.01) and earning between 91 and 290 USD on average per month (APR = 1.12, C.I = 1.02–1.23, p-value = 0.02) were more likely to have completed a given antimicrobial course as compared to those who were married/cohabiting and earned less than 15 USD respectively. The majority of participants 60% (251/418) owned animals, and 81.3% (204/251) reported using antimicrobials mainly for prevention 61.3% (125/204) or treatment of sick animals 70.6% (144/204). Among the participants, 57.4% (117/204) reported not having sold or consumed animal products within a week after exposure to antimicrobials. Interventions to prevent AMR should adopt a One Health approach to address the gap in knowledge and practices relating to the use of antimicrobials in humans and animals

Scaling-up interventions for strengthening antimicrobial stewardship using a one health approach in Wakiso District, Uganda

We implemented a multidisciplinary project between Uganda and the UK aimed at strengthening antimicrobial stewardship (AMS) in Wakiso district, with a focus on capacity building, stakeholder engagement, and knowledge exchange using a One Health approach. Project activities included: trainings and workshops on antimicrobial resistance (AMR), AMS, infection prevention and control (IPC); Global Point Prevalence Survey (GPPS) data collection and analysis; and the mentorship of lower level health facilities. Our project demonstrated that AMS interventions using a One Health approach can enhance understanding of the prudent use of antimicrobials and improve practices at health facilities and within communities

Antimicrobial stewardship in private pharmacies in Wakiso district, Uganda: a qualitative study

Background: Private pharmacies are the first point of contact for the public regarding acquisition of medicines and other pharmaceuticals in many low- and middle-income countries including Uganda. Most antimicrobial stewardship (AMS) programmes in Uganda have targeted pharmacies in public health facilities, with little known about private pharmacies. This study explored knowledge and practices related to AMS in private pharmacies in Wakiso district, central Uganda. Methods: This was a qualitative study that involved 31 in-depth interviews to explore AMS among retail private pharmacy staff including pharmacists, pharmacy technicians/dispensers, and nurses. Participants were asked about antimicrobial resistance (AMR) and AMS practices at their pharmacy. The audio-recorded interviews were transcribed verbatim and imported to NVivo 2020 (QSR International) for thematic analysis. Results: Five major themes emerged from the study: commonly sold antimicrobials; knowledge on AMR and AMS; potential contributors to AMR; practices related to AMS; and challenges to AMS. The commonly sold antimicrobials in the pharmacies with or without prescriptions were oral azithromycin, Ampiclox® (ampicillin and cloxacillin), amoxicillin, ciprofloxacin, Septrin® (co-trimoxazole), metronidazole, Flucamox® (amoxicillin and flucloxacillin), Augmentin® (amoxicillin and clavulanic acid), cephalexin, doxycycline, and chloramphenicol. Participants had heard about AMR but not AMS, although only a few correctly defined AMR. Lack of knowledge among health workers and local communities; the overuse, misuse, and abuse of antimicrobials such as non-adherence to dosage; self-medication; and purchase of drugs without prescription were identified as potential accelerators to the emergence of AMR. Current practices related to AMS in private pharmacies were limited to meetings, antimicrobial dispensing, providing client advice, record keeping, and monitoring of drugs. Cost of healthcare, client satisfaction and retention, outdated guidelines, and the business orientation of pharmacies were the main challenges related to AMS. Conclusion: There was poor knowledge of AMR and AMS, and limited AMS practices in private pharmacies. Private pharmacies have the potential to contribute to Uganda’s fight against AMR if motivated and equipped with adequate knowledge to enhance their practices related to AMS

Histone deacetylases as new therapy targets for platinum-resistant epithelial ovarian cancer

Introduction: In developed countries, ovarian cancer is the fourth most common cancer in women. Due to the nonspecific symptomatology associated with the disease many patients with ovarian cancer are diagnosed late, which leads to significantly poorer prognosis. Apart from surgery and radiotherapy, a substantial number of ovarian cancer patients will undergo chemotherapy and platinum based agents are the mainstream first-line therapy for this disease. Despite the initial efficacy of these therapies, many women relapse; therefore, strategies for second-line therapies are required. Regulation of DNA transcription is crucial for tumour progression, metastasis and chemoresistance which offers potential for novel drug targets. Methods: We have reviewed the existing literature on the role of histone deacetylases, nuclear enzymes regulating gene transcription. Results and conclusion: Analysis of available data suggests that a signifant proportion of drug resistance stems from abberant gene expression, therefore HDAC inhibitors are amongst the most promising therapeutic targets for cancer treatment. Together with genetic testing, they may have a potential to serve as base for patient-adapted therapies