Dietary isoacids effects on growth, nitrogen, and energy digestibility, and fecal VFA and microbial ecology in finishing pigs.

Isoacids are branched ketoacids which when fed to ruminants have been shown to enhance the growth of fiber-digesting organisms. Ninety finishing gilts were individually fed dietary treatments consisting of diet type: corn-soybean meal (CSBM), a diet containing 40% distillers dried grains with solubles (DDGS), or a diet containing 40% sugar beet pulp (SBP); in combination with either no feed additive (CNT), the addition of 0.50% isobutyrate (IB), or the addition of a 0.88% mix of isobutyrate, isovalerate, and 2-methylbutyrate (MX). Gilts consumed an average of 2.171 kg/d over the 28-d trial. On d 26, fresh fecal samples were collected for determination of apparent total tract digestibility (ATTD) of gross energy (GE) and nitrogen (N), determination of fecal volatile fatty acids (VFA), and evaluation of microbial ecology. There was no interaction between diet type and isoacid addition, and no main effect of isoacid or diet type on alpha or Shannon microbial diversity measures (P > 0.05). There was no interaction between isoacid addition and diet type, and no main effect of isoacid addition on microbial beta diversity (P > 0.05), but differences were observed in microbial beta diversity due to diet type (P ≤ 0.05). There was no interaction between diet type and isoacid addition observed in fecal VFA concentrations (P > 0.05), with only minor differences in fecal VFA concentrations noted due to isoacid addition (P ≤ 0.05). The interaction between diet type and isoacid addition on ATTD of dietary GE and N (P ≤ 0.01) was largely because the addition of IB did not affect ATTD of GE or N in pigs fed the CSBM diet, but increased ATTD of GE and N in pigs fed diets containing DDGS and decreased the ATTD of GE and N in pigs fed diets containing SBP. In contrast, adding a blend of isoacids (i.e., MX) reduced the ATTD of GE and N, regardless of diet type. There was no interaction between diet type and isoacid addition, and no effect of isoacid addition was observed on pig performance (P > 0.05). Diet type did not affect ADG (P > 0.05), but pigs fed diets containing DDGS or SBP consumed less feed (P = 0.01) and exhibited greater GF ratios compared to pigs fed the low-fiber CSBM diet (P ≤ 0.05). In conclusion, there was little to no effect of isoacid addition on microbial ecology, fecal VFA concentrations, ATTD of GE or N, or pig performance, but the improvement in ATTD of GE and N in pigs fed diets containing DDGS when IB was added warrants further investigation.

Tonsils at Telethon: developing a standardised collection of tonsil photographs for group A streptococcal (GAS) research.

Introduction: Group A streptococcus (GAS) infections, such as pharyngitis and impetigo, can lead to rheumatic fever and rheumatic heart disease (RHD). Australian Aboriginal and Torres Strait Islander populations experience high rates of RHD and GAS skin infection, yet rates of GAS pharyngitis are unclear. Anecdotally, clinical presentations of pharyngitis, including tonsillar hypertrophy and sore throat, are uncommon. This study aimed to develop a standardised set of tonsil photographs and determine tonsil size distribution from an urban paediatric population. Methods: A prospective cohort of children aged 3-15 years were recruited at the public events "Discover Day" and "Telethon Weekend" (October 2017) in Perth, Western Australia, Australia. Tonsil photographs, symptomatology, and GAS rapid antigen detection tests (RADT) were collected. Tonsil size was graded from the photographs using the Brodsky Grading Scale of tonsillar hypertrophy (Brodsky) by two independent clinicians, and inter-rater reliability calculated. Pharyngitis symptoms and GAS RADT were correlated, and immediate results provided. Results: Four hundred and twenty-six healthy children participated in the study over three days. The median age was seven years [interquartile range (IQR) 5.9-9.7 years]. Tonsil photographs were collected for 92% of participants, of which 62% were rated as good-quality photographs and 79% were deemed of adequate quality for assessment by both clinicians. When scored by two independent clinicians, 57% received the same grade. Average Brodsky grades (between clinicians) were 11%, 35%, 28%, 22% and 5% of grades 0,1,2,3 and 4, respectively. There was moderate agreement in grading using photographs, and minimal to weak agreement for signs of infection. Of 394 participants, 8% reported a sore throat. Of 334 GAS RADT performed, <1% were positive. Discussion: We report the first standardised use of paediatric tonsil photographs to assess tonsil size in urban-living Australian children. This provides a proof of concept from an urban-living cohort that could be compared with children in other settings with high risk of GAS pharyngitis or rheumatic fever such as remote-living Australian Indigenous populations.

Collaborative Recognition of Wellbeing Needs: A Novel Approach to Universal Psychosocial Screening on the Neonatal Unit.

Universal screening for the psychological needs of families in neonatal care is internationally recommended, but is not routinely practiced in the United Kingdom (UK). The present quality improvement project explores the clinical and operational feasibility of a novel approach to universal screening on a neonatal intensive care unit in the UK. The approach to screening taken adopts collaborative, strengths-based and dialogical methods for recognising the psychological needs of families whose baby is in hospital. A novel screening tool, developed through consultation with families, is described. Over one month, 42 out of 80 eligible families engaged with the screening protocol either at admission to the unit, transition to the special care nursery within the unit, or discharge home, with completion rates higher at admission than discharge. This led to an eightfold increase in the number of families accessing targeted or specialist psychological intervention compared to the period prior to this pilot. This project demonstrates the need for adequate capacity in the workforce to carry out a screening programme and to respond to the needs identified.

Effects of dietary protein level on intestinal function and inflammation in nursery pigs.

High crude protein (CP; 21% to 26%) diets fed during the first 21 to 28 d postweaning are viewed negatively because of a perceived increase in the incidence rates of diarrhea due to increased intestinal protein fermentation and/or augmented enteric pathogen burden. This is thought to antagonize nursery pig health and growth performance. Therefore, our objective was to evaluate the impact of low vs. high dietary CP on 21-day postweaned pig intestinal function. Analyzed parameters included ex vivo intestinal barrier integrity (ileum and colon), ileal nutrient transport, tissue inflammation, and fecal DM. One hundred and twenty gilts and barrows (average body weight) were randomly assigned to one of two diets postweaning. Diets were fed for 21 d, in two phases. Phase 1 diets: low CP (17%) with a 1.4% standardized ileal digestible (SID) Lys (LCP), or high CP (24%) with a 1.4% SID Lysine (HCP). Phase 2: LCP (17%) and a 1.35% SID lysine, or HCP (24%) formulated to a 1.35% SID lysine. Pig growth rates, feed intakes, and fecal consistency did not differ (P > 0.05) due to dietary treatment. Six animals per treatment were euthanized for additional analyses. There were no differences in colonic epithelial barrier function as measured by transepithelial electrical resistance (TER) and fluorescein isothiocyanate (FITC)-dextran transport between treatments (P > 0.05). Interleukins (IL)-1α, IL-1ß, IL-1ra, IL-2 IL-4, IL-6, and IL-12 were not different between treatments (P > 0.05). However, IL-8 and IL-18 were higher in HCP- vs. LCP-fed pigs (P < 0.05). There were no differences in fecal dry matter (DM; P > 0.05) between treatments. In the ileum, there was a tendency (P = 0.06) for TER to be higher in HCP-fed pigs, suggesting a more robust barrier. Interestingly, glucose and glutamine transport were decreased in HCP- vs. LCP-fed pigs (P < 0.05). FITC-dextran transport was not different between treatments (P > 0.05). There were also no differences in ileal cytokine concentrations between diets (P > 0.05). Taken together, the data show that low CP does not negatively impact colonic barrier function, fecal DM, or inflammation. In contrast, ileal barrier function and nutrient transport were altered, suggesting a regional effect of diet on overall intestinal function.

High dietary crude protein (CP) is thought to antagonize nursery pig enteric health. Feeding high CP diets to nursery pigs did not exacerbate intestinal health or inflammation, and overall, protein level in the diet has little impact on animal health and performance.

Specialty pharmacy: Incorporating workflow management and medication access into pharmacy lecture and laboratory courses.

BACKGROUND AND PURPOSE: The purpose of this study was to implement and evaluate a specialty pharmacy workshop across pharmacy management and skills lab courses. EDUCATIONAL ACTIVITY AND SETTING: A specialty pharmacy workshop was developed and implemented. The lecture cohort (fall 2019) consisted of a 90-min lecture in pharmacy management. The lecture/lab cohort (fall 2020) consisted of the lecture plus a 30-min pre-lab video assignment and a two-hour laboratory activity. At the completion of lab, students presented findings virtually to specialty pharmacists. Pre-surveys and post-surveys assessed knowledge (10 items), self-confidence (9 items), and attitudes (11 items). FINDINGS: Of the 123 students enrolled in the course, 88 students (71.5%) completed pre- and post-surveys. On a 10-point scale, knowledge improved from 5.6 (SD = 1.5) to 6.5 (SD = 2.0) points in the lecture cohort and from 6.0 (SD = 1.6) to 7.3 (SD = 2.0) points in the lecture/lab cohort with a significance difference favoring the lecture/lab cohort. Perceived confidence improved for five out of nine items in the lecture cohort but improved significantly for all nine items in the lecture/lab cohort. Attitudes toward learning about specialty pharmacy were generally positive for both cohorts. SUMMARY: The specialty pharmacy workshop exposed students to workflow management and medication access processes. Students perceived the workshop to be a relevant and meaningful, allowing them to feel confident in developing knowledge and understanding of specialty pharmacy topics. The workshop can be replicated at a larger scale with schools of pharmacy utilizing the integration between didactic and laboratory courses.

Soluble corn fiber, resistant corn starch, and protected butyrate effects on performance, gastrointestinal volatile fatty acids, and apparent total-tract digestibility of calcium and phosphorus in nursery pigs.

An experiment was conducted to determine how feeding calcium (Ca)-deficient diet would affect gastrointestinal pH and volatile fatty acids (VFAs), Ca digestibility, bone mineral density (BMD), and performance in nursery pigs; and if supplementation of nondigestible oligosaccharides would affect these same parameters. In total, 240 weaned pigs (BW = 7.1 kg) were placed into 80 pens with 3 pigs/pen. The eight dietary treatments consisted of: 1) positive control (PC, 0.83% total Ca), 2) negative control (NC, 0.50% total Ca), 3 and 4) NC + 5% or 7.5% soluble corn fiber (SCF), 5 and 6) NC + 5% or 7.5% resistant corn starch (rCS), 7 and 8) NC + 0.25% or 0.50% fat-protected butyrate (pBRT). Pigs were ad libitum fed the dietary treatments for 21 d to determine average daily gain (ADG), average daily feed intake (ADFI) and gain:feed ratio (GF) with a fecal sample collected from each pen to determine Ca digestibility using acid insoluble ash as the dietary marker, with 1 pig/pen euthanized on d 21 for collection of ileal and colon contents and the left humerus. Pigs fed the NC diet had a lower colonic pH compared with pigs fed the PC (P = 0.06) but no effect on total VFA was observed (P > 0.10). Pigs fed diets containing SCF and rCS had lower colonic pH and total VFA compared to pigs fed the NC diet (P ≤ 0.05). Pigs fed diets containing pBRT had greater colonic total VFA compared to pigs fed the NC diet (P ≤ 0.07), but no difference in colonic pH was observed (P > 0.10). Pigs fed the NC diet had a greater Ca digestibility compared to pigs fed the PC (P ≤ 0.01), with no treatment to the NC having any effect on Ca digestibility compared to pigs fed the NC (P > 0.10). There was no effect of dietary Ca level on BMD and no overall addition of feeding SCF, rCS, or pBRT on BMD compared to pigs fed the NC (P > 0.10). There was no impact on pig ADG, ADFI, or GF by reducing dietary Ca by 40% (i.e., pigs fed the NC) compared to pigs fed the PC (P > 0.10). Relative to pigs fed the NC, there was no overall effect of SCF, rCS, or pBRT on ADG, ADFI, or GF (P > 0.10). In conclusion, feeding young pigs a Ca-deficient diet reduced colonic pH, increased digestibility of Ca, but had no impact on bone mineralization or overall pig performance. Supplementation of nondigestible oligosaccharides pr protected butyrate had either no effect or an inconsistent effect on colonic pH, Ca, or PHOS digestibility, bone mineralization, or overall pig performance.

Calcium (Ca) is a major component of the skeleton in addition to being essential for growth and is imperative for bone mass development. Improvement in Ca absorption in Ca-deficient diets has been shown in human and rodent studies when nondigestible oligosaccharides have been consumed due to a modification of gastrointestinal conditions which increase mineral solubility. Because swine have been shown to be an excellent model for human nutrition research, an experiment was conducted to determine how a moderately Ca-deficient diet would affect gastrointestinal fermentation conditions, Ca and phosphorus (PHOS) digestibility, bone mineralization, and growth performance in nursery pigs; and if supplementation of nondigestible oligosaccharides would affect these same parameters. Results indicate that feeding young pigs a diet below recommended levels of Ca reduced colonic pH, increased apparent total-tract digestibility of Ca and PHOS, but had no impact on bone mineralization or overall pig performance. Supplementation of nondigestible oligosaccharides had inconsistent effects on colonic pH, and did not affect Ca or PHOS digestibility, bone mineralization, or overall pig performance.

Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria.

The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5' leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S), in a manner that requires initiator fMet-tRNAMet binding. In addition, we demonstrate that in human cells and in vitro, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Furthermore, we show that mtIF2 is indispensable for mitochondrial protein synthesis. Our results demonstrate an important evolutionary divergence of the mitochondrial translation system and further our fundamental understanding of a process central to eukaryotic metabolism.

PANTHER: AZD8931, inhibitor of EGFR, ERBB2 and ERBB3 signalling, combined with FOLFIRI: a Phase I/II study to determine the importance of schedule and activity in colorectal cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a therapeutic target to which HER2/HER3 activation may contribute resistance. This Phase I/II study examined the toxicity and efficacy of high-dose pulsed AZD8931, an EGFR/HER2/HER3 inhibitor, combined with chemotherapy, in metastatic colorectal cancer (CRC). METHODS: Treatment-naive patients received 4-day pulses of AZD8931 with irinotecan/5-FU (FOLFIRI) in a Phase I/II single-arm trial. Primary endpoint for Phase I was dose limiting toxicity (DLT); for Phase II best overall response. Samples were analysed for pharmacokinetics, EGFR dimers in circulating exosomes and Comet assay quantitating DNA damage. RESULTS: Eighteen patients received FOLFIRI and AZD8931. At 160 mg bd, 1 patient experienced G3 DLT; 160 mg bd was used for cohort expansion. No grade 5 adverse events (AE) reported. Seven (39%) and 1 (6%) patients experienced grade 3 and grade 4 AEs, respectively. Of 12 patients receiving 160 mg bd, best overall response rate was 25%, median PFS and OS were 8.7 and 21.2 months, respectively. A reduction in circulating HER2/3 dimer in the two responding patients after 12 weeks treatment was observed. CONCLUSIONS: The combination of pulsed high-dose AZD8931 with FOLFIRI has acceptable toxicity. Further studies of TKI sequencing may establish a role for pulsed use of such agents rather than continuous exposure. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number: NCT01862003.

Effects of short-chain fatty acids on intestinal function in an enteroid model of hypoxia.

The healthy GI tract is physiologically hypoxic, but this may be perturbed by certain acute and chronic stressors that reduce oxygen availability systemically. Short-chain fatty acids have been shown to have beneficial effects on intestinal barrier function and inflammation. Therefore, our objective was to see whether short-chain fatty acids (SCFA) would improve GI barrier function, reduce production of pro-inflammatory cytokines, and increase the expression of genes regulating GI barrier function in enteroids exposed to hypoxia. Human duodenal enteroid monolayers were placed under hypoxia (1.0% O2) for 72 h with either 24, or 48 h pre-treatment with a high acetate ratio of SCFA's or high butyrate ratio or placed under hypoxia concurrently. Transepithelial electrical resistance (TEER) increased with SCFA pre-treatment, especially 48 h of pre-treatment and this was maintained through the first 48 h of hypoxia while cells saw barrier function dramatically decrease by 72 h of hypoxia exposure. Inflammatory protein secretion largely decreased with exposure to hypoxia, regardless of SCFA pre-treatment. Gene expression of several genes related to barrier function were decreased with exposure to hypoxia, and with concurrent and 24 h SCFA pre-treatment. However, 48 h SCFA pre-treatment with a high butyrate ratio increased expression of several metabolic and differentiation related genes. Overall, pre-treatment or concurrent treatment with SCFA mixtures were not able to overcome the negative impacts of hypoxia on intestinal function and cells ultimately still cannot be sustained under hypoxia for 72 h. However, 48 h pre-treatment maintains TEER for up to 48 h of hypoxia while upregulating several metabolic genes.

ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3' phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3' phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2.

Evaluation of digestively resistant or soluble fibers, short- and medium-chain fatty acids, trace minerals, and antibiotics in nonchallenged nursery pigs on performance, digestibility, and intestinal integrity.

Three experiments (EXP) were conducted to determine the effect of feed additives on performance, intestinal integrity, gastrointestinal volatile fatty acids (VFA), and energy and nutrient digestion in nonchallenged nursery pigs. In EXP 1, 480 pigs (6.36-kg body weight, BW) were placed into 96 pens with 5 pigs/pen, and allotted to 1 of 10 dietary treatments: 1) negative control containing no feed additive (NC), 2) NC + 44 mg chlortetracycline and 38.5 mg tiamulin/kg diet (CTsb), 3) NC + 5% resistant potato starch (RSpo), 4) NC + 5% soluble corn fiber (SCF), 5) NC + 5% sugar beet pulp (SBP), 6) NC + 0.30% fatty acid mix (FAM), 7) NC + 0.10% phytogenic blend of essential oils and flavoring compounds (PHY), 8) NC + 50 mg Cu and 1,600 mg zinc oxide/kg diet (CuZn), 9) NC + 5% resistant corn starch (RScn), and 10) NC + 0.05% ß-glucan (BG) for 28 d. There was no impact of dietary treatment on BW gain or feed intake (P ≥ 0.22). Pigs fed diets containing SCF, CTsb, and RSpo resulted in microbial community differences compared to pigs fed the NC (P < 0.05). In EXP 2, 48 barrows (12.8 kg BW) were selected at the end of EXP 1 and fed the same dietary treatments they had previously received: 1) NC, 2) NC + 5% RScn, 3) NC + 5% SCF, and 4) NC + FAM for 8 d. There was no effect of feeding diets containing RScn, SCF, or FAM on in vivo intestinal permeability (P ≤ 0.21). Ileal or colon pH, concentrations of VFA did not differ due to dietary treatment (P ≥ 0.36), but pigs fed diets containing FAM resulted in a greater butyric acid concentration in the cecum compared to pigs fed the NC (P ≤ 0.05). In EXP 3, 156 pigs (6.11 kg BW) were placed into 52 pens with 3 pigs/pen and allotted to 1 of 4 dietary treatments arranged in a factorial manner: 1) NC, 2) NC + 5% RSpo, 3) NC + 0.30% FAM, and 4) NC + 5% RSpo + 0.30% FAM for 24 d. Feeding pigs diets containing RSpo did not affect BW gain (P = 0.91) while pigs fed diets containing FAM grew improved BW gain (P = 0.09). Colonic butyric acid concentrations were greater in pigs fed diets containing RSpo (P = 0.03), while pigs fed diets containing FAM exhibited reduced total VFA concentrations (P = 0.11). The results indicate that supplementing diets with digestively resistant but fermentable fibers, short- and medium-chain fatty acids, or antibiotics do not have a consistent effect, positive or negative, on markers of intestinal integrity or barrier function, intestinal VFA patterns, ATTD of energy and nutrients, or on pig performance.

In-feed antimicrobials have been an important technology in swine production for protecting health and supporting growth. However, with legislative restrictions on the use of most antibiotics for growth promotion, research is needed to evaluate in-feed additives in replacing this growth promoting technology. Thus, strategies to enhance energy and nutrient digestibility, intestinal function and integrity, gastrointestinal volatile fatty acid concentrations, and microbial ecology in nursery pigs are desirable targets. The results of the three experiments conducted herein do not indicate that supplementing diets with digestively resistant but fermentable fibers, short-medium-chain fatty acids, or antibiotics have a consistent positive or negative effect on markers of intestinal integrity or barrier function, VFA patterns (ileal, cecal, or colon), ATTD of energy and nutrients, or pig performance.

Human iPSC colon organoid function is improved by exposure to fecal fermentates.

The host-microbe interaction is critical for intestinal homeostasis. By-products from microbial metabolism of unabsorbed dietary components have been studied increasingly as potential contributors to health and disease. In vitro fermentation systems provide a way to simulate microbial activity and by-product production of the colon using human fecal samples. Objectives of the study were to determine how clarified supernatants from two different fermentation conditions affect markers of cell proliferation, differentiation, barrier function, and immune function in a human-induced pluripotent (iPSC) colon organoid model. SCFA and BCFA's of the supernatants were analyzed and were similar to known in vivo concentrations. Molecular results showed 25% of the clarified supernatant from batch fermentation led to a more physiological intestinal phenotype including increased markers of differentiation, including alkaline phosphatase, chromogranin A, SCFA transport monocarboxylate transporter-1, (6.2-fold, 2.1-fold, and 1.8-fold, respectively; p < 0.05). Mucin production (mucin-2, mucin-4) was increased in cells treated with 25% supernatant, as observed by confocal microscopy. In addition, increased tight junction expression (claudin-3) was noted by immunofluorescence in 25% supernatant- treated cells. A dose-response increase in barrier function was observed over the 72-h time course, with a twofold increase in transepithelial electrical resistance (TER) in the 25% group compared to the control group (p < 0.05). To further investigate host effects, clarified supernatants from a continuous multistage fermentation representing the ascending (AC), transverse (TC), and descending (DC) colonic domains were utilized and some regional differences were observed including increased markers of inflammation (IL-1ß, 6.15 pg/ml; IL-6, 27.58 pg/ml; TNFα, 4.49 pg/ml; p < 0.05) in DC-treated samples only. Overall, clarified supernatants represent a valuable model to examine effects of microbial by-products on host intestinal development and function and future efforts will be designed to further understand microbial communities and metabolites, along with additional host response measures.

Enhanced System Robustness of Asynchronous BCI in Augmented Reality Using Steady-State Motion Visual Evoked Potential.

This study evaluated the effect of change in background on steady state visually evoked potentials (SSVEP) and steady state motion visually evoked potentials (SSMVEP) based brain computer interfaces (BCI) in a small-profile augmented reality (AR) headset. A four target SSVEP and SSMVEP BCI was implemented using the Cognixion AR headset prototype. An active (AB) and a non-active background (NB) were evaluated. The signal characteristics and classification performance of the two BCI paradigms were studied. Offline analysis was performed using canonical correlation analysis (CCA) and complex-spectrum based convolutional neural network (C-CNN). Finally, the asynchronous pseudo-online performance of the SSMVEP BCI was evaluated. Signal analysis revealed that the SSMVEP stimulus was more robust to change in background compared to SSVEP stimulus in AR. The decoding performance revealed that the C-CNN method outperformed CCA for both stimulus types and NB background, in agreement with results in the literature. The average offline accuracies for W = 1 s of C-CNN were (NB vs. AB): SSVEP: 82% ±15% vs. 60% ±21% and SSMVEP: 71.4% ± 22% vs. 63.5% ± 18%. Additionally, for W = 2 s, the AR-SSMVEP BCI with the C-CNN method was 83.3% ± 27% (NB) and 74.1% ±22% (AB). The results suggest that with the C-CNN method, the AR-SSMVEP BCI is both robust to change in background conditions and provides high decoding accuracy compared to the AR-SSVEP BCI. This study presents novel results that highlight the robustness and practical application of SSMVEP BCIs developed with a low-cost AR headset.

Expression of SARS-CoV-2 entry factors, electrolyte, and mineral transporters in different mouse intestinal epithelial cell types.

Angiotensin-converting enzyme 2 (ACE2) and transmembrane proteases (TMPRSS) are multifunctional proteins required for SARS-CoV-2 infection or for amino acid (AA) transport, and are abundantly expressed in mammalian small intestine, but the identity of the intestinal cell type(s) and sites of expression are unclear. Here we determined expression of SARS-CoV-2 entry factors in different cell types and then compared it to that of representative AA, electrolyte, and mineral transporters. We tested the hypothesis that SARS-CoV-2, AA, electrolyte, and mineral transporters are expressed heterogeneously in different intestinal cell types by making mouse enteroids enriched in enterocytes (ENT), goblet (GOB), Paneth (PAN), or stem (ISC) cells. Interestingly, the expression of ACE2 was apical and modestly greater in ENT, the same pattern observed for its associated AA transporters B0 AT1 and SIT1. TMPRSS2 and TMPRSS4 were more highly expressed in crypt-residing ISC. Expression of electrolyte transporters was dramatically heterogeneous. DRA, NBCe1, and NHE3 were greatest in ENT, while those of CFTR and NKCC1 that play important roles in secretory diarrhea, were mainly expressed in ISC and PAN that also displayed immunohistochemically abundant basolateral NKCC1. Intestinal iron transporters were generally expressed higher in ENT and GOB, while calcium transporters were expressed mainly in PAN. Heterogeneous expression of its entry factors suggests that the ability of SARS-CoV-2 to infect the intestine may vary with cell type. Parallel cell-type expression patterns of ACE2 with B0 AT1 and SIT1 provides further evidence of ACE2's multifunctional properties and importance in AA absorption.

Dual Systems for Enhancing Control of Protein Activity through Induced Dimerization Approaches.

To reveal the underpinnings of complex biological systems, a variety of approaches have been developed that allow switchable control of protein function. One powerful approach for switchable control is the use of inducible dimerization systems, which can be configured to control activity of a target protein upon induced dimerization triggered by chemicals or light. Individually, many inducible dimerization systems suffer from pre-defined dynamic ranges and overwhelming sensitivity to expression level and cellular context. Such systems often require extensive engineering efforts to overcome issues of background leakiness and restricted dynamic range. To address these limitations, recent tool development efforts have explored overlaying dimerizer systems with a second layer of regulation. Albeit more complex, the resulting layered systems have enhanced functionality, such as tighter control that can improve portability of these tools across platforms.

A modular tool to query and inducibly disrupt biomolecular condensates.

Dynamic membraneless compartments formed by protein condensates have multifunctional roles in cellular biology. Tools that inducibly trigger condensate formation have been useful for exploring their cellular function, however, there are few tools that provide inducible control over condensate disruption. To address this need we developed DisCo (Disassembly of Condensates), which relies on the use of chemical dimerizers to inducibly recruit a ligand to the condensate-forming protein, triggering condensate dissociation. We demonstrate use of DisCo to disrupt condensates of FUS, associated with amyotrophic lateral sclerosis, and to prevent formation of polyglutamine-containing huntingtin condensates, associated with Huntington's disease. In addition, we combined DisCo with a tool to induce condensates with light, CRY2olig, achieving bidirectional control of condensate formation and disassembly using orthogonal inputs of light and rapamycin. Our results demonstrate a method to manipulate condensate states that will have broad utility, enabling better understanding of the biological role of condensates in health and disease.

Elucidating the cellular dynamics of the brain with single-cell RNA sequencing.

Single-cell RNA-sequencing (scRNA-seq) has emerged in recent years as a breakthrough technology to understand RNA metabolism at cellular resolution. In addition to allowing new cell types and states to be identified, scRNA-seq can permit cell-type specific differential gene expression changes, pre-mRNA processing events, gene regulatory networks and single-cell developmental trajectories to be uncovered. More recently, a new wave of multi-omic adaptations and complementary spatial transcriptomics workflows have been developed that facilitate the collection of even more holistic information from individual cells. These developments have unprecedented potential to provide penetrating new insights into the basic neural cell dynamics and molecular mechanisms relevant to the nervous system in both health and disease. In this review we discuss this maturation of single-cell RNA-sequencing over the past decade, and review the different adaptations of the technology that can now be applied both at different scales and for different purposes. We conclude by highlighting how these methods have already led to many exciting discoveries across neuroscience that have furthered our cellular understanding of the neurological disease.

Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly.

Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.