Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

Functional diversification of gram-negative intermembrane phospholipid transporters by intrinsic substrate preference.

The outer membrane of Gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. We investigated these functions using synthetic cold sensitivity (at 30 °C) of a strain with deletion of yhdP, but not tamB or ydbH, and fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production. Deletion of tamB, redirecting phospholipid transport to YdbH, suppresses the ΔyhdP ΔfadR cold sensitivity suggesting this phenotype is directly related to phospholipid transport. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not despite lower cardiolipin levels. In addition to increased cardiolipin, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. A parsimonious explanation for our data is that YhdP and TamB have differential substrate transport preferences, most likely with YhdP preferentially transporting more saturated phospholipids and TamB preferentially transporting more unsaturated phospholipids. We envision cardiolipin contributing to this transport preference by sterically clogging TamB-mediated transport of saturated phospholipids. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions.

Biotic countermeasures that rescue Nannochloropsis gaditana from a Bacillus safensis infection.

The natural assemblage of a symbiotic bacterial microbiome (bacteriome) with microalgae in marine ecosystems is now being investigated as a means to increase algal productivity for industry. When algae are grown in open pond settings, biological contamination causes an estimated 30% loss of the algal crop. Therefore, new crop protection strategies that do not disrupt the native algal bacteriome are needed to produce reliable, high-yield algal biomass. Bacteriophages offer an unexplored solution to treat bacterial pathogenicity in algal cultures because they can eliminate a single species without affecting the bacteriome. To address this, we identified a highly virulent pathogen of the microalga Nannochloropsis gaditana, the bacterium Bacillus safensis, and demonstrated rescue of the microalgae from the pathogen using phage. 16S rRNA amplicon sequencing showed that phage treatment did not alter the composition of the bacteriome. It is widely suspected that the algal bacteriome could play a protective role against bacterial pathogens. To test this, we compared the susceptibility of a bacteriome-attenuated N. gaditana culture challenged with B. safensis to a N. gaditana culture carrying a growth-promoting bacteriome. We showed that the loss of the bacteriome increased the susceptibility of N. gaditana to the pathogen. Transplanting the microalgal bacteriome to the bacteriome-attenuated culture reconstituted the protective effect of the bacteriome. Finally, the success of phage treatment was dependent on the presence of beneficial bacteriome. This study introduces two synergistic countermeasures against bacterial pathogenicity in algal cultures and a tractable model for studying interactions between microalgae, phages, pathogens, and the algae microbiome.

Microneedle electrochemical aptamer-based sensing: Real-time small molecule measurements using sensor-embedded, commercially-available stainless steel microneedles.

Microneedle sensors could enable minimally-invasive, continuous molecular monitoring - informing on disease status and treatment in real-time. Wearable sensors for pharmaceuticals, for example, would create opportunities for treatments personalized to individual pharmacokinetics. Here, we demonstrate a commercial-off-the-shelf (COTS) approach for microneedle sensing using an electrochemical aptamer-based sensor that detects the high-toxicity antibiotic, vancomycin. Wearable monitoring of vancomycin could improve patient care by allowing targeted drug dosing within its narrow clinical window of safety and efficacy. To produce sensors, we miniaturize the electrochemical aptamer-based sensors to a microelectrode format, and embed them within stainless steel microneedles (sourced from commercial insulin pen needles). The microneedle sensors achieve quantitative measurements in body-temperature undiluted blood. Further, the sensors effectively maintain electrochemical signal within porcine skin. This COTS approach requires no cleanroom fabrication or specialized equipment, and produces individually-addressable, sterilizable microneedle sensors capable of easily penetrating the skin. In the future, this approach could be adapted for multiplexed detection, enabling real-time monitoring of a range of biomarkers.

Relationship between selection criteria and trainee performance in medical specialty training: A retrospective longitudinal study.

BACKGROUND: Investigation of tools used for candidate selection as predictors of future performance in medical education has been primarily undertaken within the undergraduate setting, but little is known about the selection to medical specialist programs. This retrospective longitudinal study aims to explore correlations between selection tools and the performance of trainees enrolled in the Australasian College of Dermatologists (ACD), the accredited specialist medical college for training in dermatology in Australia. METHODS: Data were collected from consecutive cohorts of ACD trainees commencing from 2007 to 2015. Predictive variables were trainee demographics and selection tools (prior academic qualification; research experience; clinical experience; curriculum vitae [CV] rating; interview scores). The outcome variables were exam performance. First Year ([Pharmacology and Clinical Sciences] and Fellowship Exam [Written, Clinical, and Overall Score]). Statistical analyses included: descriptive statistics summaries; bivariate correlation of selection criteria and demographic data with exam performance; and linear regression analysis to identify predictors of exam performance. RESULTS: Demographic analysis (N = 172) showed that 64% of trainees were female, the average age was 30.5 years (± 3.47) and trainees living in high socioeconomic status (SES) areas were over-represented. Using Pearson correlation analysis, interview scores were significantly positively correlated with First Year Pharmacology results (p = 0.018), Fellowship Written results (p = 0.002), and Fellowship Overall Scores (p = 0.006). First Year Pharmacology Exam performance was most highly correlated with Fellowship Exam performance (p = 0.000). No association was identified between exam performance and gender, SES, prior academic qualification, research experience or CV rating. Linear regression analysis showed that interview score (p = 0.012), entry age (p = 0.026) and First Year Pharmacology score (p = 0.002) were predictors of Fellowship exam performance. CONCLUSION: These results suggest that the comprehensive selection tools used by ACD are effective, with interviews being a highly valid tool linked to exam performance. These findings have important practical implications for assessing the selection process in specialist dermatology training.

Practice guidelines for teledermatology in Australia.

Despite the potential of teledermatology to increase access to dermatology services and improve patient care, it is not widely practised in Australia. In an effort to increase uptake of teledermatology by Australian dermatologists and support best practice, guidelines for teledermatology for the Australian context have been developed by The University of Queensland's Centre for Online Health in collaboration with The Australasian College of Dermatologists' E-Health Committee. The guidelines are presented in two sections: 1. Guidelines and 2. Notes to support their application in practice, when feasible and appropriate. Content was last updated March 2020 and includes modalities of teledermatology; patient selection and consent; imaging; quality and safety; privacy and security; communication; and documentation and retention of clinical images. The guidelines educate dermatologists about the benefits and limitations of telehealth while articulating how to enhance patient care and reduce risk when practicing teledermatology.

A review of literature supporting the development of practice guidelines for teledermatology in Australia.

Despite the potential of teledermatology to increase access to dermatology services and improve patient care, it is not widely practised in Australia. In an effort to increase uptake of teledermatology, Australian-specific practice guidelines for teledermatology are being developed by the Australasian College of Dermatologist. This paper reports finding from literature reviews that were undertaken to inform the development of these guidelines. Results cover the following sections: Modalities of teledermatology; Patient selection and consent; Imaging; Quality and safety; Privacy and security; Communication; and Documentation and retention. The document educates providers about the benefits and limitations of telehealth while articulating how to enhance patient care and reduce risk when practicing teledermatology.

Effects of twelve weeks of capsaicinoid supplementation on body composition, appetite and self-reported caloric intake in overweight individuals.

We examined if 12 weeks of capsaicinoid (CAP) supplementation affected appetite, body composition and metabolic health markers. Seventy seven healthy male and female volunteers (30 ± 1 y, 171.2 ± 9.8 cm, 81.0 ± 2.2 kg, 27.5 ± 0.6 kg/m2) were randomly assigned to ingest either low-dose CAP (2 mg/d; L-CAP, n = 27), high-dose CAP (4 mg/d; H-CAP, n = 22) from Capsimax or placebo (corn starch; PLA, n = 28) for 12 weeks. At baseline (0 WK), 6 weeks (6 WK) and 12 weeks (12 WK) waist: hip ratio, body composition via dual energy x-ray absorptiometry (DEXA, 0 WK and 12 WK only), self-reported Calorie intakes, appetite levels via Council on Nutrition Appetite Questionnaire (CNAQ) and serum metabolic health markers (0 WK and 12 WK only) were analyzed. Moreover, an oral glucose tolerance test (OGTT) was administered at 0 WK and 12 WK, and serum glucose and insulin responses were examined 30-120 min post test-drink consumption. Waist: hip ratio significantly decreased in L-CAP from 0 WK to 6 WK (p < 0.05), although supplementation did not significantly affect body composition. H-CAP consumed less kcal/d compared to PLA at 12 WK (difference = 257 kcal/d, p < 0.05) and L-CAP participants at 12 WK (difference = 247, p < 0.05). Twenty-three percent (9/39) of the originally-enrolled H-CAP participants reported GI distress, although no participants in the L-CAP group reported such adverse events. Interestingly, H-CAP participants presented significant increases in serum insulin as well as significant decreases in serum HDL cholesterol levels from WK0 to WK12. However, supplementation did not affect the insulin response to the administered OGTT and/or other indices of insulin sensitivity. These data suggest that H-CAP supplementation reduces self-reported energy intake after 12 weeks of supplementation, and L-CAP supplementation also reduces waist: hip ratio. Longer-term effects of capsaicinoid supplementation on basal insulin and cholesterol levels warrant further investigation.

Does androgen-ablation therapy (AAT) associated autophagy have a pro-survival effect in LNCaP human prostate cancer cells?

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Androgen-ablation therapy (AAT) and chemotherapy are commonly used to treat incurable prostate cancer. To improve outcome, there is major on-going research to develop more effective treatments with less toxicity. Autophagy has been suggested from previous studies to play a potential role in cell survival and may be associated with resistance to chemotherapy. Autophagy is known to be upregulated by nutrient starvation or AAT in prostate cancer. However, its functional impact is not fully known. The present study describes the potential synergism between the blockade of autophagy and AAT alone or AAT combined with taxane chemotherapy. Hence, future combined treatment options are warranted to further investigate the clinical impact of autophagy suppression as a treatment strategy. OBJECTIVE: To study the cellular effects of the anti-androgen bicalutamide on autophagy and its potential impact on response to androgen-ablation therapy (AAT) alone or combined with docetaxel chemotherapy in human prostate cancer LNCaP cells. MATERIALS AND METHODS: LNCaP cells were treated with bicalutamide ± docetaxel, and cellular effects were assayed: lipidated LC3 (a microtubule-associated protein) for autophagy and its trafficking to fuse with lysosome; flow cytometry using propidium iodide or caspase 3 for cell death; and sulforhodamine B assay for cell growth. RESULTS: Bicalutamide treatment enhanced autophagy in LNCaP cells with increased level of autophagosome coupled with an altered cellular morphology reminiscent of neuroendocrine differentiation. Consistent with the literature on the interaction between androgen receptor activation and taxane chemotherapy, bicalutamide diminished docetaxel mediated cytotoxicity. Significantly, pharmacological inhibition of autophagy with 3-methyladenine significantly enhanced the efficacy cell kill mediated by AAT ± docetaxel. CONCLUSION: Autophagy associated with bicalutamide treatment in LNCaP cells may have a pro-survival effect and strategy to modulate autophagy may have a potential therapeutic value.

Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation.

The docking protein Gab2 is overexpressed in several human malignancies, including breast cancer, and is associated with increased metastatic potential. Here we report that Gab2 overexpression in MCF-10A mammary epithelial cells led to delayed cell spreading, a decrease in stress fibers and mature focal adhesions, and enhanced cell migration. Expression of a Gab2 mutant uncoupled from 14-3-3-mediated negative feedback (Gab2(2xA)) led to a more mesenchymal morphology and acquisition of invasive potential. Expression of either Gab2 or Gab2(2xA) led to decreased activation of RhoA, but only the latter increased levels of Rac-GTP. Expression of constitutively active RhoA in MCF-10A/Gab2 cells restored stress fibers and focal adhesions, indicating that Gab2 signals upstream of RhoA to suppress these structures. Mutation of the two Shp2-binding sites to phenylalanine (Gab2(ΔShp2)) markedly reduced the effects of Gab2 on cellular phenotype and RhoA activation. Expression of Gab2 or Gab2(2xA), but not Gab2(ΔShp2), promoted Vav2 phosphorylation and plasma membrane recruitment of p190A RhoGAP. Knockdown of p190A RhoGAP reversed Gab2-mediated effects on stress fibers and focal adhesions. The identification of a novel pathway downstream of Gab2 involving negative regulation of RhoA by p190A RhoGAP sheds new light on the role of Gab2 in cancer progression.

GRP78 up-regulation is associated with androgen receptor status, Hsp70-Hsp90 client proteins and castrate-resistant prostate cancer.

GRP78/BiP is a key member of the molecular chaperone heat shock protein (Hsp) 70 family. It has a critical role in prostate cancer (PC) including Pten loss-driven carcinogenesis, but the molecular basis of this remains unclear. We investigated the effect of GRP78 and its putative client proteins, including androgen receptor (AR) in clinical PC. Expression of GRP78 and key Hsp70-hsp90 client proteins (HER2, HER3, AR and AKT) were studied in an incidence tissue microarray (TMA) of prostate cancer. The relationship of GRP78 and AR was further tested in in vitro cell models (LNCaP and its derived LNCaP-CR subclone) and a matched TMA of hormone-naïve (HNPC) and castrate-resistant prostate cancer (CRPC). In vitro and in vivo expression of GRP78 and client proteins were assessed by western blotting and immunohistochemistry, respectively, using the weighted histoscore method. Significant co-expression of GRP78, pAKT, HER2, HER3 and AR was observed in PC. Abnormal AR, GRP78 and pAKT expression have significant impact on patient survival. GRP78 expression in AR(+) tumours was significantly higher than in AR(-) tumours. In keeping with our clinical data, activation of AR by dihydrotestosterone (DHT) potently activated GRP78 expression in both LNCaP and LNCaP-CR cells. For the first time, using a matched HNPC and CRPC TMA, enhanced cytoplasmic and membranous GRP78 expression was observed in CRPC. Future prospective studies are therefore warranted to validate GRP78 as prognostic marker and therapeutic target, in the context of the AR and pAKT status. In summary, GRP78 is co-expressed with Hsp70-hsp90 client proteins. Up-regulated expression of AR and GRP78 expression in untreated prostate cancer predicts a less favourable outcome. This points to the importance of understanding in the molecular interaction among AR, GRP78 and AKT.

Increased proliferation and altered growth factor dependence of human mammary epithelial cells overexpressing the Gab2 docking protein.

The docking protein Gab2 is a proto-oncogene product that is overexpressed in primary breast cancers. To determine the functional consequences of Gab2 overexpression, we utilized the immortalized human mammary epithelial cell line MCF-10A. In monolayer culture, expression of Gab2 at levels comparable with those detected in human breast cancer cells accelerated epidermal growth factor (EGF)-induced cell cycle progression and was associated with increased basal Stat5 tyrosine phosphorylation and enhanced and/or more sustained EGF-induced Erk and Akt activation. Three-dimensional Matrigel culture of MCF-10A cells resulted in the formation of polarized, growth-arrested acini with hollow lumina. Under these conditions, Gab2 increased cell proliferation during morphogenesis, leading to significantly larger acini, an effect dependent on Gab2 binding to Grb2 and Shp2 and enhanced by recruitment of the p85 subunit of phosphatidylinositol 3-kinase. Pharmacological inhibition of MEK revealed that, in addition to direct activation of phosphatidylinositol 3-kinase, increased Erk signaling also contributed to Gab2-mediated enhancement of acinar size. In addition, Gab2 overcame the proliferative suppression that normally occurs in late stage cultures and conferred independence of the morphogenetic program from exogenous EGF. Finally, higher levels of Gab2 expression led to the formation of large disorganized structures with defective luminal clearance. These findings support a role for Gab2 in mammary tumorigenesis.

Estrogen receptor expression in lumbosacral dorsal root ganglion cells innervating the female rat urinary bladder.

We have investigated whether bladder afferent neurons are likely to be targets for circulating estrogens by mapping estrogen receptor (ER) distribution in lumbosacral dorsal root ganglia (DRG) of adult female rats. Sensory neurons innervating either the detrusor or trigone regions were identified by application of fluorescent retrograde tracer dyes to the bladder wall. Labelled neurons were classified by their immunoreactivity for either type of ER (ERalpha or ERbeta) and further compared with subpopulations of neurons containing substance P, calcitonin gene-related peptide and vanilloid receptor (a marker of polymodal nociceptors). Both ER types were expressed in numerous sensory neurons of either upper lumbar (L1/L2) or lower lumbar/sacral (L6/S1) ganglia and there was almost complete coexpression of ERalpha and ERbeta. ER-positive neurons were mainly small-medium size (18-25-microm diameter), indicating that they may be nociceptors and/or supply visceral targets. Most bladder-projecting neurons expressed ERs and the majority of these also expressed neuropeptides or vanilloid receptor. Afferent neurons supplying detrusor and trigone regions had similar immunohistochemical features. About a third of the bladder-projecting neurons expressed both ER and vanilloid receptor, suggesting a mechanism by which estrogens could influence bladder pain. The prevalence of different chemical classes of ER-positive bladder-projecting neurons was reflected throughout the entire population of neurons in dorsal root ganglia of these spinal levels, suggesting that neurons supplying other pelvic visceral targets may have similar chemical profiles. These results suggest that many functional classes of sensory neurons innervating the lower urinary tract are likely to be targets for circulating estrogens, including many nociceptor neurons. The coexistence of ERalpha and ERbeta suggests a broad range of potential mechanisms by which estrogens may exert their genomic effects in this system.