Novel Molecular Methods for Discovery and Engineering of Biocatalysts From Uncultured Marine Microorganisms

Metagenomics is a powerful cultivation-independent approach, which can be applied to gain access to the biocatalysts from uncultured marine microorganisms. Discovery of marine biocatalysts by this approach, in general, involves four main steps. First, a metagenomic library containing a pool of biocatalyst-encoding genes is constructed from a marine environment, which can be done by various methods, including cloning of enzymatically-digested DNA, uncut DNA, and PCR-amplified products. Second, the metagenomic library is screened for the genes of interest by employing the activity assay of expression product, in situ hybridization, or Polymerase Chain Reaction (PCR). Third, the obtained target genes, both functional and phylogenetic genes, are sequenced and analysed by using bioinformatic tools in order to gain information on the functional and structural properties as well as the microbial sources of the encoded biocatalysts. Finally, the target genes are expressed in suitable microbial hosts, thereby producing the corresponding recombinant biocatalysts. All existing methods in engineering of marine biocatalysts for the performance improvement can be classified into two main strategies: (i) rational design and (ii) directed evolution. Rational design, which may include the use of resctriction enzyme(s) and splicing by overlap extension (SOE), requires information on the biocatalyst`s structural and functional properties to alter specific amino acid(s). Whereas directed evolution, including error-prone PCR technique and gene shuffling, needs no such information

Screening and Characterization of Bacterial Chitosanase From Marine Environment

Screening of extracellular chitosanase from bacterial isolates associated with marine sponges have been done. Out of 100 bacterial isolates, forty isolates were capable of forming clearing zones on the chitin media and one isolate, 34-b, produced the highest chitinolytic index. The enzymes was produced on chitin liquid medium at 37oC in a shaking waterbath for a five-day cultivation. Crude enzymes were prepared by cell-free supernatant (CFS) and concentrated through 70% (saturated) ammonium sulphate percipitation followed by dialysis. The enzymes worked best at pH and temperature of 6-7 and 60oC, respectively. The half-life (T1/2) for chitosanase activity was 500.2 min or 8.34 hours (at 37oC) and 55.12 min (at 50oC), indicating the enzyme are quite stable at that temperature. However, around 80% of the original activity was lost at 60oC after 15 min of incubation

Testing precision and accuracy of weak value measurements in an IBM quantum system

Historically, weak values have been associated with weak measurements performed on quantum systems. Over the past two decades, a series of works have shown that weak values can be determined via measurements of arbitrary strength. One such proposal by Denkmayr et al. [Phys. Rev. Lett. 118, 010402 (2017)], carried out in neutron interferometry experiments, yielded better outcomes for strong than for weak measurements. We extend this scheme and explain how to implement it in an optical setting as well as in a quantum computational context. Our implementation in a quantum computing system provided by IBM confirms that weak values can be measured, with varying degrees of performance, over a range of measurement strengths. However, at least for this model, strong measurements do not always perform better than weak ones.Comment: 9 pages, 5 figure

Microbiology clinical culture diagnostic yields and antimicrobial resistance proportions before and during the COVID-19 pandemic in an Indian community hospital and two US community hospitals

Studies comparing the impact of the COVID-19 pandemic on diagnostic microbiology culture yields and antimicrobial resistance proportions in low-to-middle-income and high-income countries are lacking. A retrospective study using blood, respiratory, and urine microbiology data from a community hospital in India and two community hospitals (Hospitals A and B) in St. Louis, MO, USA was performed. We compared the proportion of cultures positive for selected multi-drug-resistant organisms (MDROs) listed on the WHO\u27s priority pathogen list both before the COVID-19 pandemic (January 2017-December 2019) and early in the COVID-19 pandemic (April 2020-October 2020). The proportion of blood cultures contaminated with coagulase-negativ

Point Prevalence Surveys of Antimicrobial Use among Hospitalized Children in Six Hospitals in India in 2016.

The prevalence of antimicrobial resistance in India is among the highest in the world. Antimicrobial use in inpatient settings is an important driver of resistance, but is poorly characterized, particularly in hospitalized children. In this study, conducted as part of the Global Antimicrobial Resistance, Prescribing, and Efficacy in Neonates and Children (GARPEC) project, we examined the prevalence of and indications of antimicrobial use, as well as antimicrobial agents used among hospitalized children by conducting four point prevalence surveys in six hospitals between February 2016 and February 2017. A total of 681 children were hospitalized in six hospitals across all survey days, and 419 (61.5%) were prescribed one or more antimicrobials (antibacterials, antivirals, antifungals). Antibacterial agents accounted for 90.8% (547/602) of the total antimicrobial prescriptions, of which third-generation cephalosporins (3GCs) accounted for 38.9% (213/547) and penicillin plus enzyme inhibitor combinations accounted for 14.4% (79/547). Lower respiratory tract infection (LRTI) was the most common indication for prescribing antimicrobials (149 prescriptions; 24.8%). Although national guidelines recommend the use of penicillin and combinations as first-line agents for LRTI, 3GCs were the most commonly prescribed antibacterial agents (55/149 LRTI prescriptions; 36.9%). In conclusion, 61.5% of hospitalized children were on at least one antimicrobial agent, with excessive use of 3GCs. Hence there is an opportunity to limit their inappropriate use

Oxygen dependency of mitochondrial metabolism indicates outcome of newborn brain injury

There is a need for a method of real-time assessment of brain metabolism during neonatal hypoxic-ischaemic encephalopathy (HIE). We have used broadband near-infrared spectroscopy (NIRS) to monitor cerebral oxygenation and metabolic changes in 50 neonates with HIE undergoing therapeutic hypothermia treatment. In 24 neonates, 54 episodes of spontaneous decreases in peripheral oxygen saturation (desaturations) were recorded between 6 and 81 h after birth. We observed differences in the cerebral metabolic responses to these episodes that were related to the predicted outcome of the injury, as determined by subsequent magnetic resonance spectroscopy derived lactate/N-acetyl-aspartate. We demonstrated that a strong relationship between cerebral metabolism (broadband NIRS-measured cytochrome-c-oxidase (CCO)) and cerebral oxygenation was associated with unfavourable outcome; this is likely to be due to a lower cerebral metabolic rate and mitochondrial dysfunction in severe encephalopathy. Specifically, a decrease in the brain tissue oxidation state of CCO greater than 0.06 µM per 1 µM brain haemoglobin oxygenation drop was able to predict the outcome with 64% sensitivity and 79% specificity (receiver operating characteristic area under the curve = 0.73). With further work on the implementation of this methodology, broadband NIRS has the potential to provide an early, cotside, non-invasive, clinically relevant metabolic marker of perinatal hypoxic-ischaemic injury

A paleoepidemiological approach to the osteological paradox : Investigating stress, frailty and resilience through cribra orbitalia

Open Access via the Jisc Wiley Agreement Funder: British Academy (GrantNumber(s): GP2\190224)Peer reviewedPublisher PD

Suppression of HBV by Tenofovir in HBV/HIV coinfected patients : a systematic review and meta-analysis

Background: Hepatitis B coinfection is common in HIV-positive individuals and as antiretroviral therapy has made death due to AIDS less common, hepatitis has become increasingly important. Several drugs are available to treat hepatitis B. The most potent and the one with the lowest risk of resistance appears to be tenofovir (TDF). However there are several questions that remain unanswered regarding the use of TDF, including the proportion of patients that achieves suppression of HBV viral load and over what time, whether suppression is durable and whether prior treatment with other HBV-active drugs such as lamivudine, compromises the efficacy of TDF due to possible selection of resistant HBV strains. Methods: A systematic review and meta-analysis following PRISMA guidelines and using multilevel mixed effects logistic regression, stratified by prior and/or concomitant use of lamivudine and/or emtricitabine. Results: Data was available from 23 studies including 550 HBV/HIV coinfected patients treated with TDF. Follow up was for up to seven years but to ensure sufficient power the data analyses were limited to three years. The overall proportion achieving suppression of HBV replication was 57.4%, 79.0% and 85.6% at one, two and three years, respectively. No effect of prior or concomitant 3TC/FTC was shown. Virological rebound on TDF treatment was rare. Interpretation: TDF suppresses HBV to undetectable levels in the majority of HBV/HIV coinfected patients with the proportion fully suppressed continuing to increase during continuous treatment. Prior treatment with 3TC/FTC does not compromise efficacy of TDF treatment. The use of combination treatment with 3TC/FTC offers no significant benefit over TDF alone

Metagenomic Survey of Potential Symbiotic Bacteria and Polyketide Synthase Genes in an Indonesian Marine Sponge

There has been emerging evidence that the bacteria associated with marine sponges are the key producers of many complex bioactive compounds. The as-yet uncultured candidate bacterial genus “Candidatus Entotheonella” of the marine sponge Theonella swinhoei from Japan have recently been recognized as the source of numerous pharmacologically relevant polyketides and modified peptides, as previously reported by the Piel group (Wilson et al. 2014). This work reported the presence of “Candidatus Entotheonella sp.” in the highly complex microbiome of an Indonesian marine sponge from Kapoposang Island, SouthSulawesi. We further identified the Kapoposang sponge specimen used in this work as Rhabdastrella sp. based on the integrated morphological, histological, and cytochrome oxidase subunit I (COI) gene analyses. To detect the polyketide biosynthetic machinery called type I polyketide synthase (PKS) in this Indonesian Rhabdastrella sp., we amplified and cloned the ketosynthase-encoding DNA regions of approximately 700 bp from the uncultured sponge's microbiome. Further sequencing and analysis of several randomly chosen clones indicated that all of them are mostly likely involved in the biosynthesis of methyl-branched fatty acids. However, employing a PKS-targeting primer designed in this work led to the isolation of four positive clones. BlastX search and subsequent phylogenetic analysis showed that one of the positive clones, designed as RGK32, displayed high homology with ketosynthase domains of many type I PKS systems and may belong to the subclass cis-AT PKS group