Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut⁺ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio^® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L^?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016     

Uncoupling Lipid Metabolism from Inflammation through Fatty Acid Binding Protein-Dependent Expression of UCP2

Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2.     

Large fluctuations but constant mean temperatures allow corals to persist in intertidal rock pools on the east coast of South Africa

Intertidal corals have been under-studied yet they provide scope for understanding adaptation and acclimatisation of corals to marginal conditions. Corals in intertidal rock pools along the east coast of South Africa withstand large temperature fluctuations, and marginal conditions for survival and growth. Four sites along the KwaZulu-Natal (KZN) coastline were sampled to determine latitudinal differences in coral communities, from 27°S to 31°S. Water temperature of rock pools at each site was monitored to see if temperature determined coral diversity in intertidal pools. Sixteen coral species were present in rock pools overall. Each of three sites in northern and central KZN hosted 12 coral taxa whereas only six taxa occurred at the most southern site. Anomastrea irregularis was the most abundant species at all sites, followed by Pocillopora verrucosa and P. damicornis. Unexpectedly, rock pool temperatures did not show a trend with latitude and thus cannot explain this decline in coral diversity. Temperatures in isolated rock pools showed large summer day time fluctuations of more than 10 °C at spring tide. However, temperatures drop substantially at high tide, lowering the mean rock pool temperature and possibly allowing these coral communities to persist in the marginal conditions of rock pools in South Africa.     

Characterization of a Spontaneous Retinal Neovascular Mouse Model

Background

Vision loss due to vascular disease of the retina is a leading cause of blindness in the world. Retinal angiomatous proliferation (RAP) is a subgroup of neovascular age-related macular degeneration (AMD), whereby abnormal blood vessels develop in the retina leading to debilitating vision loss and eventual blindness. The novel mouse strain, neoretinal vascularization 2 (NRV2), shows spontaneous fundus changes associated with abnormal neovascularization. The purpose of this study is to characterize the induction of pathologic angiogenesis in this mouse model.

Methods

The NRV2 mice were examined from postnatal day 12 (p12) to 3 months. The phenotypic changes within the retina were evaluated by fundus photography, fluorescein angiography, optical coherence tomography, and immunohistochemical and electron microscopic analysis. The pathological neovascularization was imaged by confocal microscopy and reconstructed using three-dimensional image analysis software.

Results

We found that NRV2 mice develop multifocal retinal depigmentation in the posterior fundus. Depigmented lesions developed vascular leakage observed by fluorescein angiography. The spontaneous angiogenesis arose from the retinal vascular plexus at postnatal day (p)15 and extended toward retinal pigment epithelium (RPE). By three months of age, histological analysis revealed encapsulation of the neovascular lesion by the RPE in the photoreceptor cell layer and subretinal space.

Conclusions

The NRV2 mouse strain develops early neovascular lesions within the retina, which grow downward towards the RPE beginning at p15. This retinal neovascularization model mimics early stages of human retinal angiomatous proliferation (RAP) and will likely be a useful in elucidating targeted therapeutics for patients with ocular neovascular disease.     

The Importance of the Derivative in Sex-Hormone Cycles: A Reason Why Behavioural Measures in Sex-Hormone Studies Are So Mercurial

To study the dynamic changes in cognition across the human menstrual cycle, twenty, healthy, naturally-cycling women undertook a lateralized spatial figural comparison task on twelve occasions at approximately 3–4 day intervals. Each session was conducted in laboratory conditions with response times, accuracy rates, eye movements, salivary estrogen and progesterone concentrations and Profile of Mood states questionnaire data collected on each occasion. The first two sessions of twelve for the response variables were discarded to avoid early effects of learning thereby providing 10 sessions spread across each participant''s complete menstrual cycle. Salivary progesterone data for each participant was utilized to normalize each participant''s data to a standard 28 day cycle. Data was analysed categorically by comparing peak progesterone (luteal phase) to low progesterone (follicular phase) to emulate two-session repeated measures typical studies. Neither a significant difference in reaction times or accuracy rates was found. Moreover no significant effect of lateral presentation was observed upon reaction times or accuracy rates although inter and intra individual variance was sizeable. We demonstrate that hormone concentrations alone cannot be used to predict the response times or accuracy rates. In contrast, we constructed a standard linear model using salivary estrogen, salivary progesterone and their respective derivative values and found these inputs to be very accurate for predicting variance observed in the reaction times for all stimuli and accuracy rates for right visual field stimuli but not left visual field stimuli. The identification of sex-hormone derivatives as predictors of cognitive behaviours is of importance. The finding suggests that there is a fundamental difference between the up-surge and decline of hormonal concentrations where previous studies typically assume all points near the peak of a hormonal surge are the same. How contradictory findings in sex-hormone research may have come about are discussed.     

Molecular and morphological evidence for the Holarctic distribution of Urogonimus macrostomus (Rudolphi, 1803) Monticelli, 1888 (Digenea: Leucochloridiidae)

Species of Urogonimus Monticelli, 1888 (Leucochloridiidae Poche, 1907) are difficult to distinguish using adult morphology, and their taxonomy has been repeatedly subjected to revision. Some Nearctic species have been regarded as synonymous with the Palearctic type species Urogonimus macrostomus (Rudolphi, 1803) Monticelli, 1888. This implies that U. macrostomus is present in the Nearctic, but there is no additional evidence for this putative distribution. We collected trematodes morphologically indistinguishable from U. macrostomus from a house sparrow (Passer domesticus) in Edmonton, Alberta, Canada. Sequences 2958 bp in total length from the small and large subunits of ribosomal DNA from 2 specimens were 99.8-100% identical to those of U. macrostomus in the Ukraine and Japan. In light of the lack of morphological differences and small degree of genetic variation, we consider the specimens we collected to be conspecific with U. macrostomus in the Palearctic, and the Holarctic range of the species is thus supported. Sequences from a more rapidly evolving gene, cytochrome c oxidase 1, were obtained to aid future study of this and related species.     

Hit-to-Lead studies: the discovery of potent, orally bioavailable thiazolopyrimidine CXCR2 receptor antagonists

A Hit-to-Lead optimisation programme was carried out on a high throughput screening hit, the thiazolopyrimidine 1, resulting in the discovery of the potent, orally bioavailable CXCR2 antagonist 29.     

An Examination of Adult Women’s Sleep Quality and Sleep Routines in Relation to Pet Ownership and Bedsharing

ABSTRACT

People in many parts of the world commonly share their beds not only with human partners but also with dogs and cats. Self-report and actigraphy data have shown that sleeping with an adult human partner has both positive and negative impacts on human sleep, but there has been little exploration of the impacts that pets have on human sleep quality. We collected survey data online from 962 adult women living in the United States to investigate relationships between pet ownership and human sleep. Fifty-five percent of participants shared their bed with at least one dog and 31% with at least one cat. In addition, 57% of participants shared their bed with a human partner. Our findings did not show a strong relationship between pet ownership status or bedsharing conditions and sleep quality as assessed by the Pittsburgh Sleep Quality Index (PSQI), although according to this measure, a high percentage of study participants did experience sleep quality deficits. It is possible that pet ownership contributed to the high global PSQI scores we observed, especially since all but 7% of participants resided with dogs and/or cats. Other measures included in this study indicate that dogs and cats, and where they sleep, may indeed affect sleep habits and perceptions of sleep quality. Dog owners had earlier bedtimes and wake times than individuals who had cats but no dogs. Compared with human bed partners, dogs who slept in the owner’s bed were perceived to disturb sleep less and were associated with stronger feelings of comfort and security. Conversely, cats who slept in their owner’s bed were reported to be equally as disruptive as human partners, and were associated with weaker feelings of comfort and security than both human and dog bed partners. Follow-up research is necessary to determine if pet owners’ perceptions of pets’ impacts on their sleep align with objective measures of sleep quality.     

Reconstitution of KCNE1 into lipid bilayers: comparing the structural, dynamic, and activity differences in micelle and vesicle environments

KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.