Dual roles of tau R peptides on Cu(II)/(I)-mediated reactive oxygen species formation.

Metal dyshomeostasis plays a critical role in the reactive oxygen species (ROS) formation and protein misfolding and aggregation; hence, contributing to neurodegeneration. Tau protein plays a key role in normal cellular function by maintaining microtubule formation in brain. The role of metal ions on tau protein biochemistry has not been systematically evaluated, but earlier reports indicated that metal ions modulate the complex biochemistry of this protein and its peptides. Herein, we evaluated interactions of biologically-relevant Cu(II) ions with the four repeat peptides of tau protein (R1 through R4) and their role on the formation of ROS, Cu(II) to Cu(I) reduction, and ultimately, peptide aggregation. The role of R peptides on ROS formation was characterized in the absence and presence of biological reducing agent, ascorbate by using UV-Vis and fluorescence spectroscopy. In the presence of the reducing agent, all Cu(II)-peptide complexes reduced hydroxyl radical (OH·), while only Cu(II)-R3 complex depleted the hydrogen peroxide (H2O2). In the absence of a reducing agent, only Cu(II)-R2 and Cu(II)-R3 complexes, which contain Cys and His residues, produced OH· and H2O2. Only R2 and R3 peptides, but not R1 and R4, reduced Cu(II) to Cu(I). The aggregation propensities of R peptides were modulated by Cu(II) and ascorbate, and were imaged by transmission electron microscopy. All metallo-peptides were characterized predominantly as singly charged mononuclear complexes by mass spectrometry. The data indicate that Cu(II)-peptide complexes may act as pro-oxidants or antioxidants and exhibit unique aggregation propensities under specific environmental conditions, with implications in the biological setting.

The MEKK1 PHD ubiquitinates TAB1 to activate MAPKs in response to cytokines.

Unlike the other MAP3Ks, MEKK1 (encoded by Map3k1) contains a PHD motif. To understand the role of this motif, we have created a knockin mutant of mouse Map3k1 (Map3k1(m) (PHD)) with an inactive PHD motif. Map3k1(m) (PHD) ES cells demonstrate that the MEKK1 PHD controls p38 and JNK activation during TGF-ß, EGF and microtubule disruption signalling, but does not affect MAPK responses to hyperosmotic stress. Protein microarray profiling identified the adaptor TAB1 as a PHD substrate, and TGF-ß- or EGF-stimulated Map3k1(m) (PHD) ES cells exhibit defective non-canonical ubiquitination of MEKK1 and TAB1. The MEKK1 PHD binds and mediates the transfer of Lys63-linked poly-Ub, using the conjugating enzyme UBE2N, onto TAB1 to regulate TAK1 and MAPK activation by TGF-ß and EGF. Both the MEKK1 PHD and TAB1 are critical for ES-cell differentiation and tumourigenesis. Map3k1(m) (PHD) (/+) mice exhibit aberrant cardiac tissue, B-cell development, testis and T-cell signalling.

Effects of increasing dietary protein supplies on milk yield, milk composition, and nitrogen use efficiency in lactating buffalo.

Nitrogen efficiency of lactating buffalo can be increased by providing dietary crude protein (CP) precisely to the requirement. Twelve lactating Nili-Ravi buffaloes (6 primiparous and 6 multiparous) at 76 ± 37.5 days in milk (DIM) were used in this study. The treatments were diets providing three levels of CP (% DM basis): (1) low-protein = 11%; (2) medium-protein = 13.1%; (3) high-protein = 14.2% according to a 3 × 3 Latin square design. The period length of each treatment was 21 days and the total duration of experiment was 63 days. The diets were designed to provide similar energy. The nitrogen intake of buffalo increased linearly by increasing CP levels. Dry matter intake showed a tendency toward decrease in quadratic fashion, whereas milk yield decreased linearly in high-protein diet. No effect was observed on milk protein yield and content. Increasing the dietary CP levels increased plasma urea nitrogen, whereas glucose and triacylglycerol levels remain unaffected. Efficiency of dietary nitrogen utilization to milk averaged 21% and showed both linear and quadratic decreases by increasing the protein supply levels. In conclusion, low CP level showed higher milk production with low plasma urea nitrogen and high nitrogen efficiency in this experiment.

The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile.

Clostridium difficile infection (CDI) is an important hospital-acquired infection resulting from the germination of spores in the intestine as a consequence of antibiotic-mediated dysbiosis of the gut microbiota. Key to this is CotE, a protein displayed on the spore surface and carrying 2 functional elements, an N-terminal peroxiredoxin and a C-terminal chitinase domain. Using isogenic mutants, we show in vitro and ex vivo that CotE enables binding of spores to mucus by direct interaction with mucin and contributes to its degradation. In animal models of CDI, we show that when CotE is absent, both colonization and virulence were markedly reduced. We demonstrate here that the attachment of spores to the intestine is essential in the development of CDI. Spores are usually regarded as biochemically dormant, but our findings demonstrate that rather than being simply agents of transmission and dissemination, spores directly contribute to the establishment and promotion of disease.

Is a Carotid Doppler Scan Useful for Managing Patients with Suspected Ocular Ischemic Syndrome?

Purpose: This review aims to understand the value of a carotid Doppler scan (CDS) when managing patients with clinical/suspected ocular ischaemic syndrome (OIS); correlations between internal carotid artery (ICA) stenosis reports; subsequent patterns of referral to vascular experts; and subsequent decisions concerning surgical versus medical management. Methods: A retrospective review of 402 CDS requests by a single eye center over 4 years (2016-2019) for patients with a clinical suspicion of OIS was conducted. Data analysis included 344 patients who had reported CDS of both ICAs. We also studied referral patterns by ophthalmologists to other specialties. Results: CDS requests were related to the retina (53.2%), neuro/TIA problems (31.1%), glaucoma (10.5%) and other issues (5.2%). The majority of patients (209/344, 60.8%) had normal CDA results. Of the 688 ICAs reported, 469 (68.2%) were normal, 219 (31.8%) had atheroma present, and only 83 (12.1%) had significant stenosis. Of 83 ICAs with stenosis, 23 (27.7%) had ≥70% stenosis, 24 (28.9%) had 50-69% stenosis, and 25 (30.1%) had <50% stenosis. A total of 60/344 (17.4%) patients were referred to vascular/stroke teams: 15/60 (25%) referred had bilateral disease, and only 2/60 (3.3%) were offered carotid endarterectomy. All referred patients commenced statins and low-dose aspirin. Conclusion: Our cohort showed a low incidence of ICA stenosis according to CDS reports in patients with suspected OIS. There were very low rates of vascular and endarterectomy referral. Commencement of conservative treatment (mini aspirin+statin) by ophthalmologists could be beneficial even in the early stage of presenting clinical evidence of OIS.

Ocular ischemic syndrome (OIS) covers a wide spectrum of eye problems resulting from reduced blood flow to the eyes. OIS is commonly known to be a rapidly blinding disease due to late diagnosis. A high index of suspicion can lead to early investigation and perhaps prevent blindness with timely intervention. The fluorescein angiogram is a reliable eye test to confirm OIS disease affecting the retina. If reduced retina perfusion is confirmed, a carotid Doppler artery scan (CDS) is the next investigation to detect blood vessel lumen narrowing primarily affecting carotid arteries (neck arteries). The presence of carotid artery disease can indicate risk of stroke; hence, confirmed carotid artery disease merits a referral to vascular surgeons to consider carotid artery surgery aiming to unblock the artery and improve blood flow and hopefully reverse OIS. Our study aimed to investigate the prevalence of suspected OIS patients referred for carotid Doppler scans, correlations between carotid artery stenosis results and clinical OIS, and subsequent offers of carotid artery surgery versus conservative medical management. Our study showed that carotid artery disease severity defined by CDS has a poor correlation with clinical diagnosis of OIS. Conservative treatment is advised for all patients with carotid artery disease, whereas surgical options for carotid stenosis are rarely offered. Hence, this study questions the benefit of pursuing CDS tests in OIS patients, since the results do not change their management. Finally, we highlight the need for better guidance on carotid artery stenosis referral for carotid surgery.

Cystic Fibrosis: Understanding Cystic Fibrosis Transmembrane Regulator Mutation Classification and Modulator Therapies.

A common life-threatening hereditary disease, Cystic Fibrosis (CF), affects primarily Caucasian infants. High sweat-salt levels are observed as a result of a single autosomal mutation in chromosome 7 that affects the critical function of the cystic fibrosis transmembrane regulator (CFTR). For establishing tailored treatment strategies, it is important to understand the broad range of CFTR mutations and their impacts on disease pathophysiology. This study thoroughly investigates the six main classes of classification of CFTR mutations based on their functional effects. Each class is distinguished by distinct molecular flaws, such as poor protein synthesis, misfolding, gating defects, conduction defects, and decreased CFTR expression at the apical membrane. Furthermore, this paper focuses on the emerging field of CFTR modulators, which intend to restore CFTR function or mitigate its consequences. These modulators, which are characterized by the mode of action and targeted mutation class, have the potential to provide personalized therapy regimens in CF patients. This review provides valuable insights into the genetic basis of CF pathology, and highlights the potential for precision medicine methods in CF therapy by thoroughly investigating CFTR mutation classification and related modulators.

Effects of prilled fat supplementation in diets with varying protein levels on production performance of early lactating Nili Ravi Buffaloes.

OBJECTIVE: The objective of the current study was to find out the independent and interactive effects of prilled fat supplementation with protein on the production performance of early lactating Nili Ravi buffaloes. METHODS: Sixteen early lactating buffaloes (36.75±5.79 d in milk; mean±standard error) received 4 treatments in 4×4 Latin-square design according to 2×2 factorial arrangements. The dietary treatments were: i) low protein low fat, ii) low protein high fat, iii) high protein low fat, and iv) high protein high fat. The dietary treatments contained 2 protein (8.7% and 11.7% crude protein) and fat levels (2.6% and 4.6% ether extract) on a dry matter basis. RESULTS: The yields of milk and fat increased with increasing protein and fat independently (p≤0.05). Energy-, protein-, and fat-corrected milk yields also increased with increasing protein and fat independently (p≤0.05). Increasing dietary protein increased the protein yield by 3.75% and lactose yield by 3.15% and increasing dietary fat supplies increased the fat contents by 3.93% (p≤0.05). Milk yield and fat-corrected milk to dry matter intake ratios were increased at high protein and high fat levels (p≤0.05). Milk nitrogen efficiency was unaffected by dietary fat (p>0.10), whereas it decreased with increasing protein supplies (p≤0.05). Plasma urea nitrogen and cholesterol were increased by increasing protein and fat levels, respectively (p≤0.05). The values of predicted methane production reduced with increasing dietary protein and fat. CONCLUSION: It is concluded that prilled fat and protein supplies increased milk and fat yield along with increased ratios of milk yield and fat-corrected milk yields to dry matter intake. However, no interaction was observed between prilled fat and protein supplementation for production parameters, body weight, body condition score and blood metabolites. Predicted methane production decreased with increasing protein and fat levels.

Drawing is an important tool to learn context-based histology in an integrated undergraduate medical curriculum.

Objectives: To determine if learning histology by drawing is superior to learning by looking through a microscope only. Methods: Second year MBBS students were divided by simple random sampling into Groups A and B. Each group comprised 50 students. This mixed-methods study was conducted in an 8-week module. For the first 4 weeks, students in Group A learned histology by drawing, whereas Group B learned by seeing the text and microscopic images. For the last 4 weeks, groups were swapped by crossover design. The impact of learning by drawing was assessed by multiple choice question (MCQ) test I and test II at the end of 4 and 8 weeks, respectively. Statistical analyses of the data were conducted with SPSS version 23. The scores obtained in test I and test II were analyzed by the independent samples t-test. The paired samples t-test was applied to scores obtained by the same subject when they learned with drawing and no drawing strategies. To assess the impact of drawing on learning histology, a focus group study was conducted in six participants selected by purposive sampling. Responses to the semi-structured interview questions were analyzed by qualitative research techniques of coding, categorizing, and generation of themes. Results: The independent samples t-test showed that there was no statistically significant difference in the mean scores obtained by Groups A and B in test I and test II. However, there was a statistically significant difference when the subject learned histology by drawing compared to no drawing, as shown by the paired samples t-test. The results from the focus group study revealed that drawing had a positive impact on knowledge retention and understanding the basic concepts of histology for its application in the clinical context. Conclusion: Drawing-based learning in histology helps with the application of basic knowledge in the clinical context.

Developing whole cell standards for the microbiome field.

BACKGROUND: Effective standardisation of the microbiome field is essential to facilitate global translational research and increase the reproducibility of microbiome studies. In this study, we describe the development and validation of a whole cell reference reagent specific to the gut microbiome by the UK National Institute for Biological Standards and Control. We also provide and test a two-step reporting framework to allow microbiome researchers to quickly and accurately validate choices of DNA extraction, sequencing, and bioinformatic pipelines. RESULTS: Using 20 strains that are commonly found in the gut, we developed a whole cell reference reagent (WC-Gut RR) for the evaluation of the DNA extraction protocols commonly used in microbiome pipelines. DNA was first analysed using the physicochemical measures of yield, integrity, and purity, which demonstrated kits widely differed in the quality of the DNA they produced. Importantly, the combination of the WC-Gut RR and the three physicochemical measures allowed us to differentiate clearly between kit performance. We next assessed the ability of WC-Gut RR to evaluate kit performance in the reconstitution of accurate taxonomic profiles. We applied a four-measure framework consisting of Sensitivity, false-positive relative abundance (FPRA), Diversity, and Similarity as previously described for DNA reagents. Using the WC-Gut RR and these four measures, we could reliably identify the DNA extraction kits' biases when using with both 16S rRNA sequencing and shotgun sequencing. Moreover, when combining this with complementary DNA standards, we could estimate the relative bias contributions of DNA extraction kits vs bioinformatic analysis. Finally, we assessed WC-Gut RR alongside other commercially available reagents. The analysis here clearly demonstrates that reagents of lower complexity, not composed of anaerobic and hard-to-lyse strains from the gut, can artificially inflate the performance of microbiome DNA extraction kits and bioinformatic pipelines. CONCLUSIONS: We produced a complex whole cell reagent that is specific for the gut microbiome and can be used to evaluate and benchmark DNA extractions in microbiome studies. Used alongside a DNA standard, the NIBSC DNA-Gut-Mix RR helps estimating where biases occur in microbiome pipelines. In the future, we aim to establish minimum thresholds for data quality through an interlaboratory collaborative study. Video Abstract.

Developing standards for the microbiome field.

BACKGROUND: Effective standardisation of methodologies to analyse the microbiome is essential to the entire microbiome community. Despite the microbiome field being established for over a decade, there are no accredited or certified reference materials available to the wider community. In this study, we describe the development of the first reference reagents produced by the National Institute for Biological Standards and Control (NIBSC) for microbiome analysis by next-generation sequencing. These can act as global working standards and will be evaluated as candidate World Health Organization International Reference Reagents. RESULTS: We developed the NIBSC DNA reference reagents Gut-Mix-RR and Gut-HiLo-RR and a four-measure framework for evaluation of bioinformatics tool and pipeline bias. Using these reagents and reporting system, we performed an independent evaluation of a variety of bioinformatics tools by analysing shotgun sequencing and 16S rRNA sequencing data generated from the Gut-Mix-RR and Gut-HiLo-RR. We demonstrate that key measures of microbiome health, such as diversity estimates, are largely inflated by the majority of bioinformatics tools. Across all tested tools, biases were present, with a clear trade-off occurring between sensitivity and the relative abundance of false positives in the final dataset. Using commercially available mock communities, we investigated how the composition of reference reagents may impact benchmarking studies. Reporting measures consistently changed when the same bioinformatics tools were used on different community compositions. This was influenced by both community complexity and taxonomy of species present. Both NIBSC reference reagents, which consisted of gut commensal species, proved to be the most challenging for the majority of bioinformatics tools tested. Going forward, we recommend the field uses site-specific reagents of a high complexity to ensure pipeline benchmarking is fit for purpose. CONCLUSIONS: If a consensus of acceptable levels of error can be agreed on, widespread adoption of these reference reagents will standardise downstream gut microbiome analyses. We propose to do this through a large open-invite collaborative study for multiple laboratories in 2020. Video Abstract.

Application of Continuous Culture for Assessing Antibiotic Activity Against Mycobacterium tuberculosis.

There is a proportion of the M. tuberculosis population that is refractory to the bactericidal action of antituberculosis antibiotics due to phenotypic tolerance. This tolerance can be impacted by environmental stimuli and the subsequent physiological state of the organism. It may be the result of preexisting populations of slow growing/non replicating bacteria that are protected from antibiotic action. It still remains unclear how the slow growth of M. tuberculosis contributes to antibiotic resistance and antibiotic tolerance. Here, we present a method for assessing the activity of antibiotics against M. tuberculosis using continuous culture, which is the only system that can be used to control bacterial growth rate and study the impact of slow or fast growth on the organism's response to antibiotic exposure.

T-Cell-Specific Deletion of Map3k1 Reveals the Critical Role for Mekk1 and Jnks in Cdkn1b-Dependent Proliferative Expansion.

MAPK signaling is important for T lymphocyte development, homeostasis, and effector responses. To better understand the role of Mekk1 (encoded by Map3k1) in T cells, we conditionally deleted Map3k1 in Lck(Cre/+)Map3k1(f/f) mice, and these display larger iNKT cell populations within the liver, spleen, and bone marrow. Mekk1 signaling controls splenic and liver iNKT cell expansion in response to glycolipid antigen. Lck(Cre/+)Map3k1(f/f) mice have enhanced liver damage in response to glycolipid antigen. Mekk1 regulates Jnk activation in iNKT cells and binds and transfers Lys63-linked poly-ubiquitin onto Carma1. Map3k1 is critical for the regulation of p27(Kip1) (encoded by Cdkn1b).