Characterization of casein and alpha lactalbumin of African elephant (Loxodonta africana) milk

The current research reports partial characterization of the caseins and alpha-lactalbumin (alpha-LA) of the African elephant with proposed unique structure-function properties. Extensive research has been carried out to understand the structure of the casein micelles. Crystallographic structure elucidation of caseins and casein micelles is not possible. Consequently, several models have been developed in an effort to describe the casein micelle, specifically of cow milk. Here we report the characterization of African elephant milk caseins. The kappa-caseins and beta-caseins were investigated, and their relative ratio was found to be approximately 1:8.5, whereas alpha-caseins were not detected. The gene sequence of beta-casein in the NCBI database was revisited, and a different sequence in the N-terminal region is proposed. Amino acid sequence alignment and hydropathy plots showed that the kappa-casein of African elephant milk is similar to that of other mammals, whereas the beta-casein is similar to the human protein, and displayed a section of unique AA composition and additional hydrophilic regions compared with bovine caseins. Elephant milk is destabilized by 62% alcohol, and it is speculated that the beta-casein characteristics may allow maintenance of the colloidal nature of the casein micelle, a role that was previously only associated with K-casein. The oligosaccharide content of milk was reported to be low in dairy animals but high in some other species such as humans and elephants. In the milk of the African elephant, lactose and oligosaccharides both occur at high levels. These levels are typically related to the content of alpha-LA in the mammary gland and thus point to a specialized carbohydrate synthesis, where the whey protein alpha-LA plays a role. We report the characterization of African elephant alpha-LA. Homology modeling of the alpha-LA showed that it is structurally similar to crystal structures of other mammalian species, which in turn may be an indication that its functional properties, such as lactose synthesis, should not be impaired

Prolonged administration of beta-lactam antibiotics - a comprehensive review and critical appraisal

Prolonged infusion of β-lactam antibiotics as either extended (over at least 2 hours) or continuous infusion is increasingly applied in intensive care units around the world in an attempt to optimise treatment with this most commonly used class of antibiotics, whose effectiveness is challenged by increasing resistance rates. The pharmacokinetics of β-lactam antibiotics in critically ill patients is profoundly altered secondary to an increased volume of distribution and the presence of altered renal function, including augmented renal clearance. This may lead to a significant decrease in plasma concentrations of β-lactam antibiotics. As a consequence, low pharmacokinetic/pharmacodynamic (PK/PD) target attainment, which is described as the percentage of time that the free drug concentration is maintained above the minimal inhibitory concentration (MIC) of the causative organism (fT>MIC), has been documented for β-lactam treatment in these patients when using standard intermittent bolus dosing, even for the most conservative target (50% fT>MIC). Prolonged infusion of β-lactams has consistently been shown to improve PK/PD target attainment, particularly in patients with severe infections. However, evidence regarding relevant patient outcomes is still limited. Whereas previous observational studies have suggested a clinical benefit of prolonged infusion, results from two recent randomised controlled trials of continuous infusion versus intermittent bolus administration of β-lactams are conflicting. In particular, the larger, double-blind placebo-controlled randomised controlled trial including 443 patients did not demonstrate any difference in clinical outcomes. We believe that a personalised approach is required to truly optimise β-lactam treatment in critically ill patients. This may include therapeutic drug monitoring with real-time adaptive feedback, rapid MIC determination and the use of antibiotic dosing software tools that incorporate patient parameters, dosing history, drug concentration and site of infection. Universal administration of β-lactam antibiotics as prolonged infusion, even if supported by therapeutic drug monitoring, is not yet ready for "prime time", as evidence for its clinical benefit is modest. There is a need for prospective randomised controlled trials that assess patient-centred outcomes (e.g. mortality) of a personalised approach in selected critically ill patients including prolonged infusion of β-lactams compared with the current standard of care

Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants

A broadband cavity-enhanced spectrometer for atmospheric trace gas measurements and Rayleigh scattering cross sections in the cyan region (470–540 nm)

An incoherent broadband cavity-enhanced absorption spectroscopy (IBBCEAS) instrument for quantification of atmospheric trace gases that absorb in the cyan region of the electromagnetic spectrum (470 to 540&thinsp;nm), including NO2 and I2, is described. The instrument uses a light-emitting diode coupled to a 1&thinsp;m optical cavity consisting of a pair of mirrors in stable resonator configuration. Transmitted light is monitored using a grating spectrometer and charge-coupled device array detector. The average mirror reflectivity was determined from the N2∕He and Ar∕He ratios of scattering coefficients and was ∼99.98&thinsp;% at its maximum, yielding an effective optical path length of 6.3&thinsp;km. Cross sections of N2, O2, air, Ar, CO2, and CH4 scattering and of O4 absorption were measured and agree with literature values within the measurement uncertainty. Trace gas mixing ratios were retrieved using the spectral fitting software DOASIS (DOAS intelligent system) from 480 to 535&thinsp;nm. Under laboratory conditions, the 60&thinsp;s, 1σ measurement precisions were ±124 and ±44&thinsp;pptv for NO2 and I2, respectively. The IBBCEAS instrument sampled ambient air in Ucluelet, BC, Canada, in July 2015. IBBCEAS retrievals agreed with independent measurements of NO2 by blue diode laser cavity ring-down spectroscopy (r2=0.975), but ambient I2 concentrations were below the detection limit.</p

Distinct immune evasion in APOBEC‐enriched, HPV‐negative HNSCC

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (n = 496) and a validation set (DKTK MASTER cohort, n = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately reanalyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy

Pathogenic Connexin-31 Forms Constitutively Active Hemichannels to Promote Necrotic Cell Death

Mutations in Connexin-31 (Cx31) are associated with multiple human diseases including erythrokeratodermia variabilis (EKV). The molecular action of Cx31 pathogenic mutants remains largely elusive. We report here that expression of EKV pathogenic mutant Cx31R42P induces cell death with necrotic characteristics. Inhibition of hemichannel activity by a connexin hemichannel inhibitor or high extracellular calcium suppresses Cx31R42P-induced cell death. Expression of Cx31R42P induces ER stress resulting in reactive oxygen species (ROS) production, in turn, to regulate gating of Cx31R42P hemichannels and Cx31R42P induced cell death. Moreover, Cx31R42P hemichannels play an important role in mediating ATP release from the cell. In contrast, no hemichannel activity was detected with cells expressing wildtype Cx31. Together, the results suggest that Cx31R42P forms constitutively active hemichannels to promote necrotic cell death. The Cx31R42P active hemichannels are likely resulted by an ER stress mediated ROS overproduction. The study identifies a mechanism of EKV pathogenesis induced by a Cx31 mutant and provides a new avenue for potential treatment strategy of the disease