Optimization of Batch Production of Bifidobacterium Pseudocatenulatum G4 in A Milk-Based Medium

This study was undertaken to optimize the production of probiotic in a milk-based medium and to establish process parameters for the batch cultivation of Bifidobacterium pseudocatenulatum G4. The locally isolated strain exhibited high tolerance to pH 1.0-3.0 and fulfilled other probiotic criteria. Identification of the organism was done using polymerase chain reaction (PCR) based method. A defined band at 1.35 kb and 289 bp were produced using genus-specific and species-specific 16S rRNA primers, respectively. An initial screening of bacteria were done using 23 full factorial design in order to identify the effect of medium components consisting of skim milk, yeast extract and glucose towards biomass production. Results showed that yeast extract had a significant positive effect on viable cell count whereas glucose resulted in a negative effect, which was then eliminated from the study. Response surface methodology (RSM) was then applied to optimize the use of skim milk and yeast extract. A quadratic model was derived using a face-centered central composite design to represent cell mass as a function of the two variables. The optimized medium composition of 2.8% (w/v) skim milk and 2.2% (w/v) yeast extract gave the maximum biomass concentration of 1.3 x 109 cfu mL-1, which was 3 log unit higher compared to the commonly used 10.0% (w/v) skim milk (6.3 x 105 cfu mL-1). The application of RSM resulted in an improvement in biomass production in a more cost-effective medium, where the skim milk composition was reduced by 71.8%. Further improvement on the biomass production was carried out in a 2-L stirred tank bioreactor. The highest viable cell count was obtained at pH 6.5, with 0.56 ms-1 impeller tip speed. Scaling-up fermentation to a 10-L stirred tank bioreactor based on constant impeller tip speed (0.56 ms-1) successfully yielded reproducible fermentation kinetic values. The results were similar to the smaller-scale reactor. Under this condition, the following were obtained: maximum biomass concentration, Xmax (1.4 x 109 cfu mL-1), maximum specific growth rate, μmax (0.48 h-1), biomass productivity, Px (7.70 x 107 cfu mL-1 h-1), and biomass yield, Yx/s (9.46 x 1010 cfu g lactose-1). The survival of B. pseudocatenulatum G4 during freeze-drying and spray-drying processes was also evaluated. During freeze-drying, the strain exhibited high percentage survival (71.7 - 82.1%) when different combinations of skim milk and sugar solutions (glucose, sucrose and lactose) were used as cryoprotectants. The viable cell counts of 2.1 x 109 cfu g-1 to 3.1 x 109 cfu g-1 were obtained after the lyophilization process. Since the addition of sugar did not result in higher percentage survival, 10.0% (w/v) skim milk was suggested as a suitable cryoprotectant. On the other hand, the strain experienced over 99.0% loss in viability after spray-drying regardless of the spray-drier air outlet temperature and use of heat-adaptation treatments

Penicillium marneffei Infection in AIDS

Penicillium marneffei is a dimorphic fungus which is endemic in Southeast Asia. It is an opportunistic pathogen which has emerged to become an AIDS-defining illness in the endemic areas. Early diagnosis with prompt initiation of treatment is crucial for its management. Prompt diagnosis can often be established through careful cytological and histological examination of clinical specimens although microbiological culture remains the gold standard for its diagnosis. Standard antifungal treatment for AIDS patients with penicilliosis is well established. Highly active antiretroviral therapy should be started early together with the antifungal treatment. Special attention should be paid to potential drug interaction between antiretroviral and antifungal treatments. Secondary prophylaxis may be discontinued with a low risk of relapse of the infection once the immune dysfunction has improved

Identifying Mechanisms of Resistance by Circulating Tumor DNA in EVOLVE, a Phase II Trial of Cediranib Plus Olaparib for Ovarian Cancer at Time of PARP Inhibitor Progression

Circulating tumor; Cediranib; Ovarian cancerADN tumoral circulante; Cediranib; Cáncer de ovarioADN tumoral circulant; Cediranib; Càncer d'ovariPurpose: To evaluate the use of blood cell–free DNA (cfDNA) to identify emerging mechanisms of resistance to PARP inhibitors (PARPi) in high-grade serous ovarian cancer (HGSOC). Experimental Design: We used targeted sequencing (TS) to analyze 78 longitudinal cfDNA samples collected from 30 patients with HGSOC enrolled in a phase II clinical trial evaluating cediranib (VEGF inhibitor) plus olaparib (PARPi) after progression on PARPi alone. cfDNA was collected at baseline, before treatment cycle 2, and at end of treatment. These were compared with whole-exome sequencing (WES) of baseline tumor tissues. Results: At baseline (time of initial PARPi progression), cfDNA tumor fractions were 0.2% to 67% (median, 3.25%), and patients with high ctDNA levels (>15%) had a higher tumor burden (sum of target lesions; P = 0.043). Across all timepoints, cfDNA detected 74.4% of mutations known from prior tumor WES, including three of five expected BRCA1/2 reversion mutations. In addition, cfDNA identified 10 novel mutations not detected by WES, including seven TP53 mutations annotated as pathogenic by ClinVar. cfDNA fragmentation analysis attributed five of these novel TP53 mutations to clonal hematopoiesis of indeterminate potential (CHIP). At baseline, samples with significant differences in mutant fragment size distribution had shorter time to progression (P = 0.001). Conclusions: Longitudinal testing of cfDNA by TS provides a noninvasive tool for detection of tumor-derived mutations and mechanisms of PARPi resistance that may aid in directing patients to appropriate therapeutic strategies. With cfDNA fragmentation analyses, CHIP was identified in several patients and warrants further investigation

Safety evaluation of Bifidobacterium pseudocatenulatum G4 as assessed in BALB/c mice

Aims:  To assess the safety of Bifidobacterium pseudocatenulatum G4 in BALB/c mice that involves examination of bacterial translocation, changes in the internal organs and histology of the intestinal lining. Methods and Results:  Forty male BALB/c mice were randomly assigned into five groups (n = 8). Three groups were orally fed with 50 μl of three different concentrations of B. pseudocatenulatum G4 (2 × 104, 1 × 108 and 1 × 1011 CFU day−1) for 4 weeks. One group was orally administered with 50 μl of 1 × 108 CFU B. longum BB536 per day for 4 weeks and last group was used as a nonbifidobacterial treatment control, which received 50 μl of skim milk. The administered strains did not affect the general health of mice and incapable of carrying out translocation to blood or liver. There were no significant differences in the internal organ (liver, heart, kidney and spleen) indices, serum enzymes of liver (aspartate aminotransferase, alkaline phosphate, alanine aminotransferase) and kidney (urea and creatinine) and histology (villi height, crypts height, mucosa thickness and epithelial cell height) of caecum, ileum and colon. Conclusion:  Administration of high dose of up to 1 × 1011 CFU B. pseudocatenulatum G4 per day to mice did not show any health threatening symptoms. Significance and Impact of the Study: Bifidobacterium pseudocatenulatum G4 is none pathogenic to BALB/c mice and could be safe probiotic for human consumption

Viability of bifidobacterium pseudocatenulatum G4 after spray-drying and freeze-drying

Viability of Bifidobacterium pseudocatenulatum G4 following spray-drying and freeze-drying in skim milk was evaluated. After spray-drying, the strain experienced over 99% loss in viability regardless of the air outlet temperature (75 and 85°C) and the heat-adaptation temperature (45 and 65 °C, 30 min). The use of heat-adaptation treatment to improve the thermotolerance of this strain was ineffective. On the other hand, the strain showed a superior survival at 71.65%–82.07% after freeze-drying. Viable populations of 9.319–9.487 log10 cfu/g were obtained when different combinations of skim milk and sugar were used as cryoprotectant. However, the addition of sugars did not result in increased survival during the freeze-drying process. Hence, 10% (w/v) skim milk alone is recommended as a suitable protectant and drying medium for this strain. The residual moisture content obtained was 4.41% ± 0.44%

Evaluation of self-efficacy-based intervention: improving school food handlers’ selected food safety behavior

Food safety training of food handlers is commonly used to reduce the incidence of foodborne disease worldwide. Nevertheless, studies have shown that the provision of knowledge alone may not necessarily result in a positive behavior change. Thus, this study aimed to determine the effect of a multiple-component intervention approach, comprising training and a self-efficacy building program, to improve hand washing and contamination prevention behavior among food handlers at public school canteens in Malaysia. Two groups were compared: treatment (n = 31) and control (n = 30). The treatment group received a 2-h on-site interactive training and a self-efficacy building program that incorporated verbal persuasion, role modeling, and practice time. Both groups were measured using a survey and the direct observation method, before and after a 14-day intervention period. Results show that the intervention package produced a significant increase (P < 0.001) in the behavioral compliance, knowledge, and self-efficacy scores for both behaviors. An increase in the overall frequency of soap use and adherence to the correct hand washing technique for the treatment group was recorded, although there is room for improvement in the post intervention hand washing compliance score. Findings from this study provide valuable information on possible ways to improve food safety behavior among school food handlers

A MIQE-Compliant Real-Time PCR Assay for Aspergillus Detection

PMCID: PMC3393739This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Comparative toxicoproteogenomics of mouse and rat liver identifies TCDD-resistance genes

The aryl hydrocarbon receptor (AHR) mediates many toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, the AHR alone does not explain the widely different outcomes among organisms. To identify the other factors involved, we evaluated three transgenic mouse lines, each expressing a different rat AHR isoform (rWT, DEL, and INS) providing widely different resistance to TCDD toxicity, as well as C57BL/6 and DBA/2 mice which exhibit a similar to tenfold divergence in TCDD sensitivity (exposures of 5-1000 mu g/kg TCDD). We supplement these with whole-genome sequencing, together with transcriptomic and proteomic analyses of the corresponding rat models, Long-Evans (L-E) and Han/Wistar (H/W) rats (having a similar to 1000-fold difference in their TCDD sensitivities; 100 mu g/kg TCDD), to identify genes associated with TCDD-response phenotypes. Overall, we identified up to 50% of genes with altered mRNA abundance following TCDD exposure are associated with a single AHR isoform (33.8%, 11.7%, 5.2% and 0.3% of 3076 genes altered unique to rWT, DEL, C57BL/6 and INS respectively following 1000 mu g/kg TCDD). Hepatic Pxdc1 was significantly repressed in all three TCDD-sensitive animal models (C57BL/6 and rWT mice, and L-E rat) after TCDD exposure. Three genes, including Cxxc5, Sugp1 and Hgfac, demonstrated different AHRE-1 (full) motif occurrences within their promoter regions between rat strains, as well as different patterns of mRNA abundance. Several hepatic proteins showed parallel up- or downward alterations with their RNAs, with three genes (SNRK, IGTP and IMPA2) showing consistent, strain-dependent changes. These data show the value of integrating genomic, transcriptomic and proteomic evidence across multi-species models in toxicologic studies.Peer reviewe

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Needs assessment and development of intervention to improve food safety behavior among food handlers in selected school canteens in the Klang Valley, Malaysia

In Malaysia, the number of foodborne outbreaks in schools has increased over the years. One of the main contributing factors is the improper handling of food among food handlers. The study aimed to develop an intervention to improve two selected food safety behaviors (handwashing and preventing contamination of ready-to-eat food) among food handlers at public school canteens. An-extended Health Action Model (HAM) was used as a framework to conduct a needs assessment, using a mixed-method approach comprising of focus group, survey and direct observation. A total of seven focus group discussions with food handlers (n=64) were first carried out to identify their perceived barriers and motivation to perform handwashing and preventing food contamination, which were used as part of the instrument adaptation and validation process. This was followed by a baseline survey (n=211) on food handlers’ knowledge , belief, norm, motivation, habit, and behavioral intention according to the HAM constructs. The proposed research model extends HAM by incorporating five dimensions of beliefs (i.e., self-efficacy, perceived susceptibility, severity, barriers, and benefits) and four dimensions of motivation (law and enforcement, reward, internal motivation, and resources). Analysis using PLS-SEM (v3.0) revealed that norm and knowledge significantly influenced belief, ultimately affecting food handlers’ intention to perform handwashing practices (p≤0.001), instead, the intention-behavior relationship, specifically for contamination prevention, was not supported in this study (p>0.05). Importance and Performance Matrix Analysis (IPMA) revealed that selfefficacy was identified as a priority variable to focus on the development of the intervention. An educational intervention program focusing on enhancing self-efficacy was developed and tested using a treatment group (n=31) and a control group (n=30). The effectiveness of the intervention package was evaluated after a 14-day intervention period. The intervention program produced a significant increase (p<0.001) in the behavioral compliance, knowledge and self-efficacy scores. Even though there are rooms for improvement, the post-intervention handwashing compliance score shows an increase in the overall frequency of soap use and compliance with the correct handwashing technique among the participants. Findings from this study provide valuable information on the possible use of the HAM model to develop a customized food safety educational program to improve food safety behavior among school food handlers. This study is the first one known to test the HAM using observational food safety behavioral data empirically. Future studies should focus on identifying other variables that may bridge the intention-behaviour gap, especially for contamination prevention