Comparative transcriptomic profile analysis of fed-batch cultures expressing different recombinant proteins in Escherichia coli

There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-β), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-β and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression

Developing Tailor-Made Microbial Consortium for Effluent Remediation

The work describes a biofilm-based soluble sulphate reduction system, which can treat up to 1600 ppm of soluble sulphate within 3.5 hours of incubation to discharge level under ambient condition using a well-characterized sulphate-reducing bacterial (SRB) consortium. This system ensures the treatment of 1509 litres of sulphate solution in 24 hours using a 220-litre bioreactor. Performance of the system during series operation was compromised, indicating the presence of inhibitor in solution at a toxic level. A single unit bioreactor would be the ideal configuration for this consortium. Modified designs of bioreactors were tested for optimization of the process using response surface methodology (RSM), where the system could function optimally at an initial sulphate concentration of 1250 ppm with a flow rate of 1.8 litre/hour. The time course of sulphate reduction yielded a parabolic profile (with coefficient of determination r 2 = 0.99 and p value < 0.05). The rate of sulphate reduction was found to be independent of seasonal variation as well as the specific design characteristic

Effects of antibiotic resistance, drug target attainment, bacterial pathogenicity and virulence, and antibiotic access and affordability on outcomes in neonatal sepsis: an international microbiology and drug evaluation prospective substudy (BARNARDS)

Background Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin–gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. Methods In BARNARDS, consenting mother–neonates aged 0–60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic–pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. Findings Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin–gentamicin, ceftazidime–amikacin, piperacillin–tazobactam–amikacin, and amoxicillin clavulanate–amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime–amikacin than for neonates treated with ampicillin–gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14–0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin–gentamicin; 286 (73·3%) to amoxicillin clavulanate–amikacin; 301 (77·2%) to ceftazidime–amikacin; and 312 (80·0%) to piperacillin–tazobactam–amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin–gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate–amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime–amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin–tazobactam–amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis

An inverse metabolic engineering approach for the design of an improved host platform for over-expression of recombinant proteins in <it>Escherichia coli</it>

<p>Abstract</p> <p>Background</p> <p>A useful goal for metabolic engineering would be to generate non-growing but metabolically active quiescent cells which would divert the metabolic fluxes towards product formation rather than growth. However, for products like recombinant proteins, which are intricately coupled to the growth process it is difficult to identify the genes that need to be knocked-out/knocked-in to get this desired phenotype. To circumvent this we adopted an inverse metabolic engineering strategy which would screen for the desired phenotype and thus help in the identification of genetic targets which need to be modified to get overproducers of recombinant protein. Such quiescent cells would obviate the need for high cell density cultures and increase the operational life span of bioprocesses.</p> <p>Results</p> <p>A novel strategy for generating a library, consisting of randomly down regulated metabolic pathways in <it>E. coli</it> was designed by cloning small genomic DNA fragments in expression vectors. Some of these DNA fragments got inserted in the reverse orientation thereby generating anti-sense RNA upon induction. These anti-sense fragments would hybridize to the sense mRNA of specific genes leading to gene ‘silencing’. This library was first screened for slow growth phenotype and subsequently for enhanced over-expression ability. Using Green Fluorescent Protein (GFP) as a reporter protein on second plasmid, we were able to identify metabolic blocks which led to significant increase in expression levels. Thus down-regulating the <it>ribB</it> gene (3, 4 dihydroxy-2-butanone-4-phosphate synthase) led to a 7 fold increase in specific product yields while down regulating the gene <it>kdpD</it> (histidine kinase) led to 3.2 fold increase in specific yields.</p> <p>Conclusion</p> <p>We have designed a high throughput screening approach which is a useful tool in the repertoire of reverse metabolic engineering strategies for the generation of improved hosts for recombinant protein expression.</p

Role of nebulised dexmedetomidine, midazolam or ketamine as premedication in preschool children undergoing general anaesthesia—A prospective, double-blind, randomised study

Background and Aims: Preschool age children are psycho-biologically vulnerable to all surgical procedures. In this study, we investigated the effect of nebulised dexmedetomidine, midazolam and ketamine as sedative premedication for alleviating parental separation anxiety, facilitating face mask acceptance and reducing emergence agitation in paediatric patients undergoing general anaesthesia. Methods: A prospective, randomised, double-blind study was done involving 96 children of age 3–7 years, randomly allocated into three equal groups and pre-medicated with either nebulised dexmedetomidine 2 μg/kg (GroupD), midazolam 0.2 mg/kg (GroupM) or ketamine 2 mg/kg (Group K). The scores of sedation scale, parental separation anxiety scale, mask acceptance scale and emergence agitation scale were recorded along with haemodynamic parameters. Two-way repeated measures analysis of variance (ANOVA), post hoc test and Kruskal–Wallis test were used for statistical analysis. Results: A statistically significant difference in sedation score was seen between the different study groups, χ2(2) = 8.561, P = 0.014 with mean rank sedation score of 56.50 for Group D, 38.92 for Group M and 43.84 for Group K. Parental separation anxiety scale score and Mask acceptance scale score also showed statistically significant difference between the different study groups, χ2(2) = 9.369, P = 0.009 and χ2(2) = 11.97, P = 0.003, respectively. Conclusion: Nebulisation with dexmedetomidine produced easy parental separation, more satisfactory sedation and face mask acceptance with less postoperative agitation than nebulisation with midazolam or ketamine

Altered metabolic profile in early and late onset preeclampsia: an FTIR spectroscopic study

Objective: Metabolic anomalies, if any, between early and late onset preeclampsia [PE] were explored using Fourier transform infrared [FTIR] spectroscopy. Setting: Department of Gynecology and Obstetrics, SSKM Hospital, IPGMER, Kolkata and Midnapur Medical College Hospital, Midnapur, India. Sample: 80 pregnant women attending routine antenatal care units; (i) early onset PE [gestational age; GA &#60; 34 weeks] (ii) late onset PE [GA &#62; 34 weeks] (iii) early onset control [GA 24–34 weeks] and (iv) late onset control [GA &#62; 34 weeks]. Methods: Serum FTIR spectra were obtained in the wave-number range of 600–4000 cm−1 at 4 cm−1 resolution. 1H NMR and estimation of atherosclerotic index (AI) were performed to validate the FTIR findings. Main outcome measure(s): Clinical characteristics and metabolic profile. Results: 13 spectral peaks corresponding to the carbohydrate, protein and lipid region were significantly altered in early onset PE [P &#60; 0.001; at 95% confidence interval]. Discriminant analysis identified five highly significant wave-numbers (1078, 1088, 1122, 1169 and 1171 cm−1) having &#8805;80% overall accuracy. Hierarchical cluster analysis of the obtained spectra at these 5 wave-numbers provided excellent segregation of early and late onset PE with respect to their controls. Principal component analysis revealed that these 5 wave-numbers significantly separated the two sub-groups of PE (97.95% of the total variance). 1H NMR results showed that serum levels of glutamate, choline, alanine and lactate were significantly higher while ariginine and citrate were significantly decreased in early onset PE as compared to late onset cases. Conclusion: Our study reveals differences in metabolomic profiles of early and late onset preeclamptic cases