Secondary DNA transfer on denim using a human blood analogue

DNA quantification technology has increased in accuracy and sensitivity, now allowing for detection and profiling of trace DNA. Secondary DNA transfer occurs when DNA is deposited via an intermediary source (e.g. clothing, tools, utensils). Multiple courtrooms have now seen secondary transfer introduced as an explanation for DNA being present at a crime scene, but sparse experimental studies mean expert opinions are often limited. Here, we used bovine blood and indigo denim substrates to quantify the amount of secondary DNA transfer and quality of STRs under three different physical contact scenarios: passive, pressure, and friction. We showed that the DNA transfer was highest under a friction scenario, followed by pressure and passive treatments. The STR profiles showed a similar, albeit less pronounced trend, with correctly scored alleles and genotype completeness being highest under a friction scenario, followed by pressure and passive. DNA on the primary substrate showed a decrease in concentration and genotype completeness both immediately and at 24 hours, suggestive of a loss of DNA during the primary transfer. The majority of secondary transfer samples amplified less than 50% of STR loci regardless of contact type. This study showed that while DNA transfer is common between denim, this is not manifested in full STR profiles. We discuss the possible technical solutions to partial profiles from trace DNA, and more broadly the ubiquity of secondary DNA transfer.

Study of the composition of mango pulp and whey for lactic fermented beverages

Fermented lactic beverages have high sensory acceptance and may present probiotic microorganisms that are able to promote well-being. Milk is the primary substrate for this type of fermentation. However, more and more, consumers have been requesting for alternative formulations with lower (or absent) milk content. Two nutritional components can allow the reduction of milk in formulations: cheese whey (CW) and fruit pulps. Thus, this study aimed to investigate, with the aid of the experimental design tool and sensory analysis, the best formulations between mango pulp (MP), CW and whole milk (WM) to develop lactic beverages fermented by Lactobacillus acidophilus La-5 at 37 °C for up to 72 h. The results indicated that the increase in MP proportion associated with a decrease in WM elevated the sensory acceptance. Three formulations, with 24 h of fermentation, containing 55 – 100 % (w/w) of MP and 0 – 45 % (w/w) of CW were the best formulations and presented pH of 3.5 – 4.0, cell count >108 CFU/mL and 82 – 88 % of global acceptance. These results suggest MP or MP with CH as substrates for lactic fermentation replacing milk.

Techno-economic analysis of recombinant Endo-β-1,4-Glucanase production from Escherichia coli Eg-RK2 culture using oil palm empty fruit bunch

A techno-economic analysis of recombinant cellulase production from E. coli Eg-RK2 was conducted to support the fulfilling of Indonesia’s energy roadmap for ethanol production. The plant utilizes OPEFB as a primary substrate in cellulase production, with an expected lifetime of 12 years. The plant is assumed to be built in Indonesia and it will fulfill 1% of the total market demand. The effect of different pretreatment processes (alkaline, steam explosion, and sequential acid-alkaline) on the profitability parameter was also studied. A simulation using SuperPro Designer was used to calculate the mass and energy balance based on the kinetic parameters of E. coli EgRK2. A technology evaluation showed that alkaline pretreatment provides the highest yield with no known inhibitors formed. The steam explosion pretreatment offers the lowest rate of lignin and hemicellulose removal, and it is understood to form known fermentation inhibitors. The NPVs of the alkaline, steam explosion and sequential acid-alkaline pretreatments are USD 32,121,000, USD -36,841,000, and USD 384,000, respectively, which means the alkaline pretreatment is economically very feasible for the production of cellulase.

Role of Ascorbic Acid in the Extraction and Quantification of Potato Polyphenol Oxidase Activity

The ability to accurately measure the activity of polyphenol oxidase (PPO) in complex matrices is essential. A problem encountered when using spectrophotometric methods is interference due to ascorbic acid (AA), often used as an enzyme “protecting agent” during PPO extraction. This study focuses on the nature of AA’s effect on spectrophotometric determinations of PPO activity as well as enzyme extraction. Potato extracts and semi-purified PPO were used as enzyme sources. The inactivation of PPO attributed to AA is substrate-mediated. The extent of AA-dependent inactivation of PPO in model systems varied between substrates. AA only slows mechanism-based inactivation of PPO induced by catechol, possibly owing to the prevention of quinone formation. AA minimally protects PPO activity during enzyme extraction. The problem associated with AA in PPO assay could be circumvented by using ascorbate oxidase to remove AA when catechol is the primary substrate or by using chlorogenic acid as the primary substrate.

Monapinone Coupling Enzyme Produces Non-Natural Heterodimers

The monapinone coupling enzyme (MCE), a fungal multicopper oxidase, catalyzes the regioselective C–C coupling between tricyclic monapinone A (the primary substrate) and other monapinones (secondary substrates) to produce atropisomeric biaryl homo- or heterodimers. In this study, mono-, bi- and tricyclic compounds were tested to determine whether they worked as secondary substrates for MCE. Among 14 cyclic compounds, MCE utilized semivioxanthin, YWA1, 1,3-naphthalenediol and flaviolin as secondary substrates to produce non-natural heterodimers. The atropisomeric biaryl heterodimers produced by MCE from monapinone A and semivioxanthin were isolated, and their structures were elucidated by NMR and MS. These findings indicate that MCE recognizes bi- and tricyclic compounds with a 1,3-dihydroxy or 1-hydroxy-3-methoxy benzene ring as a secondary substrate.

Cometabolic biodegradation of chlorinated ethenes with methanotrophs in anaerobic/aerobic simulated aquifer

Aim: This study explores anaerobic/aerobic biodegradation efficiencies of aerobic cometabolism with methanotrophs when contaminants trichloroethylene (TCE) and cis-1,2-dichloroethylene (cDCE) are present individually or in tandem. Methodology: Batch tests and an anaerobic/aerobic column system were used to simulate saturated, contaminated aquifers. A brown glass bottle with an effective volume of 44 m l-1 was prepared for the batch test. An integrated one-dimensional sequential anaerobic/aerobic column system was used to simulate the accumulative intermediates such as TCE, cDCE and VC caused by incomplete degradation of PCE during the upgradient anaerobic stage in the saturated aquifer. In the downgradient aquifer, aerobic cometabolism was employed to degrade the intermediates. Methanotrophs in the aerobic aquifer were inoculated to degrade the by-products of incomplete degradation of PCE by aerobic cometabolism. Results: In the batch test, biodegradation of TCE was significantly inhibited by cDCE. However, biodegradation of cDCE was not significantly inhibited by TCE. In the simulated aquifer test, aerobic cometabolism completely degraded intermediates (TCE, cDCE, and VC) produced by incomplete anaerobic degradation of tetrachloroethylene (PCE). The results showed that methane, a by-product of anaerobic reductive dechorination of PCE, was used as a primary substrate for aerobic degradation, at a utilization rate of almost 100%. Interpretation: Biodegradation of TCE was significantly inhibited by cDCE. Bioremediation should have sufficient oxygen and methane at aerobic stage to ensure that chlorinated ethenes fully mineralize.

Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

Neuronal complexity is attenuated in preclinical models of migraine and restored by HDAC6 inhibition

Migraine is the third most prevalent disease worldwide but the mechanisms that underlie migraine chronicity are poorly understood. Cytoskeletal flexibility is fundamental to neuronal-plasticity and is dependent on dynamic microtubules. Histone-deacetylase-6 (HDAC6) decreases microtubule dynamics by deacetylating its primary substrate, α-tubulin. We use validated mouse models of migraine to show that HDAC6-inhibition is a promising migraine treatment and reveal an undiscovered cytoarchitectural basis for migraine chronicity. The human migraine trigger, nitroglycerin, produced chronic migraine-associated pain and decreased neurite growth in headache-processing regions, which were reversed by HDAC6 inhibition. Cortical spreading depression (CSD), a physiological correlate of migraine aura, also decreased cortical neurite growth, while HDAC6-inhibitor restored neuronal complexity and decreased CSD. Importantly, a calcitonin gene-related peptide receptor antagonist also restored blunted neuronal complexity induced by nitroglycerin. Our results demonstrate that disruptions in neuronal cytoarchitecture are a feature of chronic migraine, and effective migraine therapies might include agents that restore microtubule/neuronal plasticity.

Characterization of Plasmodium falciparum Pantothenate Kinase and Identification of Its Inhibitors From Natural Products

Coenzyme A (CoA) is a well-known cofactor that plays an essential role in many metabolic reactions in all organisms. In Plasmodium falciparum, the most deadly among Plasmodium species that cause malaria, CoA and its biosynthetic pathway have been proven to be indispensable. The first and rate-limiting reaction in the CoA biosynthetic pathway is catalyzed by two putative pantothenate kinases (PfPanK1 and 2) in this parasite. Here we produced, purified, and biochemically characterized recombinant PfPanK1 for the first time. PfPanK1 showed activity using pantetheine besides pantothenate, as the primary substrate, indicating that CoA biosynthesis in the blood stage of P. falciparum can bypass pantothenate. We further developed a robust and reliable screening system to identify inhibitors using recombinant PfPanK1 and identified four PfPanK inhibitors from natural compounds.