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Abstract Background Individuals with precapillary pulmonary hypertension (PH) experience severely impaired quality of life. A disease-specific outcome measure for PH, the Cambridge Pulmonary Hypertension Outcome Review (CAMPHOR) was developed and validated in the UK and subsequently adapted for use in additional countries. The aim of this study was to translate and assess the reliability and validity of the CAMPHOR for German-speaking populations. Methods Three main adaptation stages involved; translation (employing bilingual and lay panels), cognitive debriefing interviews with patients and validation (assessment of the adaptation’s psychometric properties). The psychometric evaluation included 107 patients with precapillary PH (60 females; age mean (standard deviation) 60 (15) years) from 3 centres in Austria, Germany and Switzerland. Results No major problems were found with the translation process with most items easily rendered into acceptable German. Participants in the cognitive debriefing interviews found the questionnaires relevant, comprehensive and easy to complete. Psychometric analyses showed that the adaptation was successful. The three CAMPHOR scales (symptoms, activity limitations and quality of life) had excellent test-retest reliability correlations (Symptoms = 0.91; Activity limitations = 0.91; QoL = 0.90) and internal consistency (Symptoms = 0.94; Activity limitations = 0.93; QoL = 0.94). Predicted correlations with the Nottingham Health Profile provided evidence of the construct validity of the CAMPHOR scales. The CAMPHOR adaptation also showed known group validity in its ability to distinguish between participants based on perceived general health, perceived disease severity, oxygen use and NYHA classification. Conclusions The CAMPHOR has been shown to be valid and reliable in the German population and is recommend for use in clinical practice.

Abstract Background A low resting heart rate (HR) is prognostically favourable in healthy individuals and in patients with left heart disease. In this study we investigated the impact of HR at diagnosis on long-term outcome in patients with differently classified precapillary pulmonary hypertension (pPH). Methods pPH patients diagnosed as pulmonary arterial (PAH) or inoperable chronic thromboembolic pulmonary hypertension (CTEPH) were registered and regularly followed at our centre Baseline characteristics and events defined as either death or lung transplantation were noted. The prognostic value of HR was analysed using Kaplan Meier estimates, live tables and Cox regression. Results 206 patients with PAH (148) and inoperable CTEPH (58) were included. The median HR was 82 bpm. pPH with a HR below 82 bpm had a significantly longer overall event-free survival (2409 vs.1332 days, p = .000). This advantage was similarly found if PAH and CTEPH were analysed separately. Although a lower HR was associated with a better hemodynamic and functional class, HR was a strong and independent prognostic marker for transplant free survival even if corrected for age, sex, hemodynamics and functional status. Conclusion We show that resting HR at diagnosis is a strong and independent long-term prognostic marker in PAH and CTEPH. Whether reducing HR by pharmacological agents would improve outcome in pPH has to be assessed by future trials with high attention to safety.

Abstract Background Martorell hypertensive ischemic leg ulcer (Martorell ulcer) is characterized by distinct alterations in the arteriolar wall of subcutaneous vessels, leading to progressive narrowing of the vascular lumen and increase of vascular resistance. These changes are similar to the alterations observed in pulmonary arterioles in patients with chronic pulmonary hypertension (PH). This study was aimed to assess an association between the two disorders. Methods In this case–control study, 14 patients with Martorell ulcer were clinically assessed for the presence of pulmonary hypertension using transthoracic Doppler echocardiography. Data from patients were compared to 28 matched hypertensive controls. Results Systolic pulmonary arterial pressure (sPAP) in patients with Martorell ulcer was significantly higher than in the control group (33.8 ± 16.9 vs 25.3 ± 6.5 mmHg, p = 0.023); the prevalence of pulmonary hypertension was 31% (5/14) in patients and 7% (2/28) in controls (p = 0.031). No differences were seen in left heart size and function between patients and controls. Conclusion This study provides first evidence that subcutaneous arteriolosclerosis, the hallmark of Martorell ulcer, is associated with PH. These findings suggest that patients with Martorell leg ulcer might be at significant risk to develop elevated pulmonary arterial pressure. Patients with leg ulcers who present with dyspnea should be evaluated by echocardiography for the presence of pulmonary hypertension.

Abstract Background Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and still incurable disease. Research of IPAH-pathogenesis is complicated by the lack of a direct access to the involved tissue, the human pulmonary vasculature. Various auto-antibodies have been described in the blood of patients with IPAH. The purpose of the present work was therefore to comparatively analyze peripheral blood B lymphocyte RNA expression characteristics in IPAH and healthy controls. Methods Patients were diagnosed having IPAH according to WHO (mean pulmonary arterial pressure ≥ 25 mmHg, pulmonary capillary occlusion pressure ≤ 15 mmHg, absence of another explaining disease). Peripheral blood B-lymphocytes of patients and controls were immediately separated by density gradient centrifugation and magnetic beads for CD19. RNA was thereafter extracted and analyzed by the use of a high sensitivity gene chip (Affymetrix HG-U133-Plus2) able to analyze 47000 transcripts and variants of human genes. The array data were analyzed by two different softwares, and up-and down-regulations were defined as at least 1.3 fold with standard deviations smaller than fold-changes. Results Highly purified B-cells of 5 patients with IPAH (mean pulmonary artery pressure 51 ± 13 mmHg) and 5 controls were analyzed. Using the two different analyzing methods we found 225 respectively 128 transcripts which were up-regulated (1.3–30.7 fold) in IPAH compared with healthy controls. Combining both methods, there were 33 overlapping up-regulated transcripts and no down-regulated B-cell transcripts. Conclusion Patients with IPAH have a distinct RNA expression profile of their peripheral blood B-lymphocytes compared to healthy controls with some clearly up-regulated genes. Our finding suggests that in IPAH patients B cells are activated.

Background: Early nutritional challenges can lead to permanent metabolic changes, increasing risk of developing chronic diseases later in life. Total parenteral nutrition (TPN) is a life-saving nutrition regimen, used especially in intrauterine growth-restricted (IUGR) neonates. Early TPN feeding alters metabolism, but whether these alterations are permanent is unclear. Programmed metabolism is likely caused by epigenetic changes due to imbalances of methyl nutrients., Objectives: We sought to determine whether feeding TPN in early life would increase risk of developing dyslipidemia in adulthood and whether supplementing the methyl nutrients betaine and creatine to TPN would prevent this development. We also sought to determine whether IUGR exacerbates the effects of neonatal TPN on lipid metabolism in adulthood., Methods: Female piglets (n = 32; 7 d old) were used in 4 treatments: 24 normal-weight piglets were randomly assigned to sow-fed (SowFed), standard TPN (TPN-control), and TPN with betaine and creatine (TPN-B+C); 8 IUGR piglets were fed control TPN (TPN-IUGR) as a fourth group. After 2 wk of treatment, all pigs were then fed a standard solid diet. At 8 mo old, central venous catheters were implanted to conduct postprandial fat tolerance tests., Results: Feeding TPN in the neonatal period led to dyslipidemia in adulthood, as indicated by higher postprandial triglyceride (TG) levels in TPN-control (P < 0.05), compared with SowFed. IUGR piglets were particularly sensitive to neonatal TPN feeding, as TPN-IUGR piglets developed obesity and dyslipidemia in adulthood, as indicated by greater backfat thickness (P < 0.05), higher liver TG (P < 0.05), slower postprandial TG clearance (P < 0.05), and elevated fasting plasma nonhigh-density lipoprotein-cholesterol (P < 0.01), and nonesterified fatty acids (P < 0.001), compared with TPN-control., Conclusions: Feeding TPN in early life increases the risk of developing dyslipidemia in adulthood, especially in IUGR neonates; however, methyl nutrient supplementation to TPN did not prevent TPN-induced changes in lipid metabolism., Competing Interests: Conflict of interest RFB is an Editorial Board Member for the Journal of Nutrition and played no role in the Journal’s evaluation of the manuscript. All other authors report no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11-q13. A deletion of the SNORD116 cluster, encoding small nucleolar RNAs, or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS, respectively. By investigation of their subcellular localization, we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2, a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases, we identified protein interaction partners for WT or mutant MAGEL2, in particular the survival motor neuron protein (SMN), involved in spinal muscular atrophy, and the fragile-X-messenger ribonucleoprotein (FMRP), involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism, while regulation of transcription was mainly observed for WT MAGEL2. Hence, we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus, including the SNORD116 cluster. Thereby, we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116, SNORD115, and SNORD109A, as well as protein-coding genes MKRN3 and SNRPN, thus bridging the gap between PWS and SYS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Perinatal mental health disorders (PMDs) are a global health concern. In industrialized countries, the prevalence of PMDs is estimated to be 20%, and they are associated with serious negative effects for women, their children and their families, along with high societal costs related to long-term impacts. In Switzerland, the PMD detection rate during obstetrical healthcare provision is very low (1-3%), and specialized healthcare services are limited. This study aimed to develop and implement an advanced practice midwife (APM) role at a Swiss obstetrics and gynecology hospital using the PEPPA framework to provide adequate screening and first-consultation services., Methods: The study uses a qualitative approach and follows the research stages using the 8-step from the participatory, evidence-based, patient-focused process for advanced practice nursing role development, implementation and evaluation (PEPPA) framework to develop and implement the APM role., Results: Utilizing the PEPPA framework, we were able to develop, implement, and evaluate the APM role in the field of perinatal mental health. Through appropriate screening and first-consultation services, we were able to identify affected women early and facilitate treatment., Conclusions: In addition to stakeholder engagement and interprofessional collaboration, PEPPA serves as a beneficial framework for the process of role development, implementation, and evaluation in the midwifery profession. This study aims to assist midwives with Master's degrees in establishing corresponding roles within their practice areas, thereby enhancing care delivery. Furthermore, the current APM approach is intended to be continuously evaluated to gain new insights into its effectiveness., Competing Interests: The authors have each completed and submitted an ICMJE form for Disclosure of Potential Conflicts of Interest. The authors declare that they have no competing interests, financial or otherwise, related to the current work. S. Walther reports that he has an R01 grant for research in depression from the National Institute of Health, 2 project grants for research in schizophrenia from the Swiss National Science Foundation, a grant for research in depression from the Era-Net Neuron Program, and a grant for research on suicide prevention from the Swiss Health Promotion Foundation. He also reports that he has payments or honoraria for lectures from: Mepha, Neurolite, and Lundbeck., (© 2024 Sutter L. et al.)

The goal of this project was to determine whether various measures of mammary development differed between gilts and multiparous sows at the end of gestation. During gestation, Yorkshire × Landrace gilts ( n  = 19) and sows (second and third gestations, n  = 17) were fed one daily meal of a conventional corn-based diet, where the amount fed was based on body weight (BW) and backfat thickness (BF) at mating. On day 110 ± 1 of gestation, a jugular blood sample was obtained from all gilts and sows to measure insulin-like growth factor-1 (IGF-1), glucose, free fatty acids, and urea. On that same day, BW and BF were measured and animals were euthanized. Mammary glands from one side of the udder were dissected for compositional analyses. The fifth gland of the contralateral row of mammary glands was sampled for histology and immunohistochemical localization of Ki67. There was less total parenchyma (1,437.4 vs. 2,004.7 ± 127.1 g; P  < 0.001) and total extraparenchymal tissue (1,691.0 vs. 2,407.0 ± 125.3 g; P  < 0.001) in mammary glands of gilts compared to those from sows. When these values were expressed per kg BW (226.0 and 284.0 ± 2.7 kg for gilts and sows, respectively), parenchymal mass did not differ ( P  > 0.10), while extraparenchymal tissue weight tended to be less in gilts than sows ( P  = 0.07). All components within the parenchyma differed by parity ( P  < 0.001). Specifically, parenchymal tissue from gilts contained a greater proportion of fat and dry matter (DM), a lower proportion of protein, and lower concentrations of DNA (6.59 vs. 9.35 ± 0.53 mg/g DM) and RNA (7.76 vs. 12.33 ± 0.70 mg/g DM) than that from sows. On the other hand, the circumference of alveolar lumens was greater in gilts than sows ( P  < 0.001), while the percentage of epithelial cells that were positive for Ki67, a marker of cell proliferation, was greater in sows than gilts ( P <  0.05). Circulating concentrations of IGF-1 were greater in gilts than in multiparous sows (45.0 vs. 27.3 ± 2.8 ng/mL, P  < 0.001). None of the other blood variables were changed by parity. Results show a marked effect of parity on mammary gland development in swine. At the end of gestation, the mammary glands of gilts had less parenchyma with lower epithelial proliferation than glands from multiparous sows. These differences could alter the response of mammary tissue to various nutritional or endocrine signals. This information is crucial for the development of management strategies designed to maximize sow milk yield., Competing Interests: The authors declare no conflict of interest., (© His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2024.)

Seasonally circulating viruses, such as Influenza, as well as newly emerging viruses and variants thereof, and waning immunity urge the need for safe, easy-to-use and inexpensive drugs to protect from these challenges. To prevent transmission of these viruses and subsequent excessive inflammatory reactions on mucous membranes, we tested the efficacy of the natural essence P80 as spray and in form of lozenges against respiratory infections caused by SARS-CoV-2 variants of concern (VoCs), influenza A (H3N2) and influenza B (Victoria). P80 natural essence, a Dimocarpus longan extract, shielded highly differentiated human airway epithelia from SARS-CoV-2 wildtype and Omicron variant as well as Influenza A and B infection and dampened inflammation by down-modulating pro-inflammatory cytokine and anaphylatoxin secretion. A single application of P80 natural essence spray maintained tissue integrity long-term. This also significantly reduced the release of infectious viral particles and the secretion of IP10, MCP1, RANTES and C3a, all of which mediate the migration of immune cells to the sites of infection. Even P80 lozenges dissolved in distilled water or non-neutralizing saliva efficiently prevented SARS-CoV-2 and Influenza-induced tissue destruction. Consequently, our in vitro data suggest that P80 natural essence can act as antiviral prophylactic, both in form of nasal or oral spray and in form of lozenges, independent of circulating respiratory challenges., (© 2024. The Author(s).)

Fifty-two multiparous sows (average parity 3.1 ± 0.9 and initial BW 245.6 ± 32.5 kg) were used to evaluate the effects of dietary standardized ileal digestible (SID) Lys-to-net energy (NE) ratios on nitrogen (N) utilization throughout a 24-d lactation period. Sows were randomly assigned to one of five isoenergetic feeding programs that provided equally spaced and increasing SID Lys-to-NE ratios between 2.79 and 5.50 g SID Lys/Mcal NE. The feeding programs were generated by blending the two extreme diets in varying proportions and were provided to sows immediately after farrowing (day 1) and until weaning at day 24 ± 1. Nitrogen balances were conducted between days 4 and 7, 12 and 15, and 20 and 23 ± 1 of lactation to represent weeks 1, 2, and 3, respectively, using total urine collection and fecal grab sampling. Contrast statements were used to determine the linear and quadratic effects of increasing Lys-to-NE ratios. Linear and quadratic broken-line and polynomial quadratic (QPM) models were used to determine the optimum dietary Lys-to-NE ratios for N retention in milk. The Bayesian information criterion was used to assess the best fit. Feeding program did not influence sow average daily feed intake (5.8 ± 0.1 kg), BW change (-8.2 ± 3.1 kg), or change in back fat thickness (-2.6 ± 0.7 mm) over the 24-d lactation period, but piglet average daily gain increased with dietary SID Lys-to-NE ratio (linear; P < 0.05). Sow N intake increased with increasing dietary Lys-to-NE ratio in weeks 2 and 3 (linear; P < 0.001). Whole-body N retention (N intake - N output in urine and feces) increased with increasing dietary Lys-to-NE ratio in all weeks (linear; P < 0.05). The N retention in milk tended to increase then decrease with increasing dietary Lys-to-NE ratio in weeks 1 and 2 (quadratic; P = 0.051 and P = 0.081) and the QPM showed optimal milk N retention at 4.28, 4.42, and 4.67 g Lys/Mcal NE for weeks 1, 2, and 3, respectively. Maternal N retention (N intake - N output in urine, feces, and milk) decreased and then increased in week 1 (quadratic; P < 0.01) and increased in weeks 2 and 3 (linear; P < 0.01) with increasing dietary Lys-to-NE ratio. Therefore, the SID Lys-to-NE ratio necessary to optimize milk N output is dynamic throughout lactation. A two-diet feeding program could be created to match optimal weekly or daily SID Lys-to-NE ratios, which could lead to improved piglet ADG and body weights at weaning., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Fifty gilts (initial body weight [BW] 190.7 ± 4.2 kg) were recruited on day 85 of gestation and were used until day 19 of lactation to assess the dose-response of inactivated yeast via hydrolyzation (HY) inclusion on offspring growth and immunoglobulin (Ig) transfer prior to weaning. Gilts were assigned to one of the 5 experimental diets: a control with no HY (HY0) or inclusion of 0.25% (HY0.25), 0.5% (HY0.5), 1.0% (HY1.0), or 1.2% (HY1.2) HY. Gilts were weighed on days 85 and 110 of gestation and days 1 and 19 (weaning) after farrowing. Offspring were weighed on days 1 and 19 of age. On lactation day 1 (approximately 24 h after farrowing), colostrum, gilt plasma, and plasma from 2 median BW piglets were collected and on day 19, plasma from each gilt and 2 median BW piglets per litter were collected for determination of Ig concentrations. Contrast statements were used to assess the linear, quadratic, cubic, and quartic effects of HY inclusion. The inclusion of HY had minimal effects on gilt BW or litter characteristics at birth (total number born and born alive, piglet birth weight). Lactation average daily feed intake of the gilts tended to increase then decrease with increasing HY inclusion (quadratic; P = 0.085). Piglet preweaning average daily gain (linear, quadratic, and quartic; P < 0.05) and BW at weaning (quadratic and quartic; P < 0.05) increased then decreased with increasing HY inclusion. On lactation day 1, colostrum and gilt plasma Ig concentrations were not affected by dietary treatment (P > 0.10) but piglet IgA and IgM decreased then increased with HY inclusion level (cubic; P < 0.05). On lactation day 19, piglet plasma IgG tended to increase with HY inclusion (linear; P = 0.099). In summary, increasing HY inclusion in late gestating and lactating gilt diets improved immune transfer in the first 24 h after birth and piglet preweaning growth rates and BW at weaning. Therefore, maternal feeding of HY could be used as a strategy to improve offspring immunocompetence and BW at weaning, with possible carryover benefits for the postweaning phase., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Precision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology-a strategy that is based on perturbing primary tumor cells from cancer patients-could provide a road forward to personalize treatment. We extend this paradigm to measuring proteome activity landscapes by acquiring quantitative phosphoproteomic data from patient-derived organoids (PDOs). We show that kinase inhibitors induce inhibitor- and patient-specific off-target effects and pathway crosstalk. Reconstruction of the kinase networks revealed that the signaling rewiring is modestly affected by mutations. We show non-genetic heterogeneity of the PDOs and upregulation of stemness and differentiation genes by kinase inhibitors. Using imaging mass-cytometry-based profiling of the primary tumors, we characterize the tumor microenvironment (TME) and determine spatial heterocellular crosstalk and tumor-immune cell interactions. Collectively, we provide a framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno-) oncology in CRC., Competing Interests: H.C. is an inventor on several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/. H.C. is currently head of pharma Research Early Development (pRED) at Roche. H.C. holds several patents on organoid technology. Their application numbers, followed by their publication numbers (if applicable), are as follows: PCT/NL2008/050543, WO2009/022907; PCT/NL2010/000017, WO2010/090513; PCT/IB2011/002167, WO2012/014076; PCT/IB2012/052950, WO2012/168930; PCT/EP2015/060815, WO2015/173425; PCT/EP2015/077990, WO2016/083613; PCT/EP2015/077988, WO2016/083612; PCT/EP2017/054797, WO2017/149025; PCT/EP2017/065101, WO2017/220586; PCT/EP2018/086716, n/a; and GB1819224.5, n/a., (© 2023 The Author(s).)

Introduction: To explore whether the reported lower pathogenicity in infected individuals of variant of concern (VoC) Omicron and its current subvariants compared to VoC Delta may be related to fundamental differences in the initial virus-tissue interaction, we assessed their ability to penetrate, replicate and cause damage in a human 3D respiratory model., Methods: For this, we used TEER measurements, real-time PCR, LDH, cytokine and complex confocal imaging analyses., Results and Discussion: We observed that Delta readily penetrated deep into the respiratory epithelium and this was associated with major tissue destruction, high LDH activity, high viral loads and pronounced innate immune activation as observed by intrinsic C3 activation and IL-6 release at infection sites. In contrast, Omicron subvariants BA.5, BQ.1.1 and BF7 remained superficially in the mucosal layer resulting merely in outward-directed destruction of cells, maintenance of epithelial integrity, minimal LDH activity and low basolateral release of virus at infection sites, as well as significantly smaller areas of complement activation and lower IL-6 secretion. Interestingly, also within Omicron subvariants differences were observed with newer Omicron subvariants BQ.1.1 and BF.7 illustrating significantly reduced viral loads, IL-6 release and LDH activity compared to BA.5. Our data indicate that earliest interaction events after SARS-CoV-2 transmission may have a role in shaping disease severity., Competing Interests: Author LH was a co-founder of ADSI GmbH and consults in cell biology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zaderer, Abd El Halim, Wyremblewsky, Lupoli, Dächert, Muenchhoff, Graf, Blum, Lass-Flörl, Keppler, Huber, Posch and Wilflingseder.)

Delta-like homolog 1 ( Dlk1 ), an inhibitor of adipogenesis, controls the cell fate of adipocyte progenitors. Experimental data presented here identify two independent regulatory mechanisms, transcriptional and translational, by which Ifrd1 (TIS7) and its orthologue Ifrd2 (SKMc15) regulate Dlk1 levels. Mice deficient in both Ifrd1 and Ifrd2 (dKO) had severely reduced adipose tissue and were resistant to high-fat diet-induced obesity. Wnt signaling, a negative regulator of adipocyte differentiation, was significantly upregulated in dKO mice. Elevated levels of the Wnt/β-catenin target protein Dlk1 inhibited the expression of adipogenesis regulators Pparg and Cebpa , and fatty acid transporter Cd36 . Although both Ifrd1 and Ifrd2 contributed to this phenotype, they utilized two different mechanisms. Ifrd1 acted by controlling Wnt signaling and thereby transcriptional regulation of Dlk1 . On the other hand, distinctive experimental evidence showed that Ifrd2 acts as a general translational inhibitor significantly affecting Dlk1 protein levels. Novel mechanisms of Dlk1 regulation in adipocyte differentiation involving Ifrd1 and Ifrd2 are based on experimental data presented here., Competing Interests: IV, DC, KP, TV, DH, RG, ME, IT, PE, ED, MH, VK, GD, JR, MK, MH, TV, LH No competing interests declared, (© 2023, Vietor et al.)

Congenital glucose-galactose malabsorption is a rare autosomal recessive disorder caused by mutations in SLC5A1 encoding the apical sodium/glucose cotransporter SGLT1. We present clinical and molecular data from eleven affected individuals with congenital glucose-galactose malabsorption from four unrelated, consanguineous Turkish families. Early recognition and timely management by eliminating glucose and galactose from the diet are fundamental for affected individuals to survive and develop normally. We identified novel SLC5A1 missense variants, p.Gly43Arg and p.Ala92Val, which were linked to disease in two families. Stable expression in CaCo-2 cells showed that the p.Ala92Val variant did not reach the plasma membrane, but was retained in the endoplasmic reticulum. The p.Gly43Arg variant, however, displayed processing and plasma membrane localization comparable to wild-type SGLT1. Glycine-43 displays nearly invariant conservation in the relevant structural family of cotransporters and exchangers, and localizes to SGLT1 transmembrane domain TM0. p.Gly43Arg represents the first disease-associated variant in TM0; however, the role of TM0 in the SGLT1 function has not been established. In summary, we are expanding the mutational spectrum of this rare disorder., Competing Interests: The authors declare no conflict of interest.

Background: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling., Methods: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan ( Bgn ), Tlr3 , and IFN-α/β receptor alpha chain ( Ifnar1 )-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans., Results: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn -/- , Tlr3 -/- , and Ifnar1 -/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/β receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans., Conclusions: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD., Competing Interests: Disclosures None.

Heterozygous mutations in the gene encoding RagD GTPase were shown to cause a novel autosomal dominant condition characterized by kidney tubulopathy and cardiomyopathy. We previously demonstrated that RagD, and its paralogue RagC, mediate a non-canonical mTORC1 signaling pathway that inhibits the activity of TFEB and TFE3, transcription factors of the MiT/TFE family and master regulators of lysosomal biogenesis and autophagy. Here we show that RagD mutations causing kidney tubulopathy and cardiomyopathy are "auto- activating", even in the absence of Folliculin, the GAP responsible for RagC/D activation, and cause constitutive phosphorylation of TFEB and TFE3 by mTORC1, without affecting the phosphorylation of "canonical" mTORC1 substrates, such as S6K. By using HeLa and HK-2 cell lines, human induced pluripotent stem cell-derived cardiomyocytes and patient-derived primary fibroblasts, we show that RRAGD auto-activating mutations lead to inhibition of TFEB and TFE3 nuclear translocation and transcriptional activity, which impairs the response to lysosomal and mitochondrial injury. These data suggest that inhibition of MiT/TFE factors plays a key role in kidney tubulopathy and cardiomyopathy syndrome., (© 2023. The Author(s).)

Objective: In brown adipose tissue (iBAT), the balance between lipid/glucose uptake and lipolysis is tightly regulated by insulin signaling. Downstream of the insulin receptor, PDK1 and mTORC2 phosphorylate AKT, which activates glucose uptake and lysosomal mTORC1 signaling. The latter requires the late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) complex, which serves to translate the nutrient status of the cell to the respective kinase. However, the role of LAMTOR in metabolically active iBAT has been elusive., Methods: Using an AdipoqCRE-transgenic mouse line, we deleted LAMTOR2 (and thereby the entire LAMTOR complex) in adipose tissue (LT2 AKO). To examine the metabolic consequences, we performed metabolic and biochemical studies in iBAT isolated from mice housed at different temperatures (30 °C, room temperature and 5 °C), after insulin treatment, or in fasted and refed condition. For mechanistic studies, mouse embryonic fibroblasts (MEFs) lacking LAMTOR 2 were analyzed., Results: Deletion of the LAMTOR complex in mouse adipocytes resulted in insulin-independent AKT hyperphosphorylation in iBAT, causing increased glucose and fatty acid uptake, which led to massively enlarged lipid droplets. As LAMTOR2 was essential for the upregulation of de novo lipogenesis, LAMTOR2 deficiency triggered exogenous glucose storage as glycogen in iBAT. These effects are cell autonomous, since AKT hyperphosphorylation was abrogated by PI3K inhibition or by deletion of the mTORC2 component Rictor in LAMTOR2-deficient MEFs., Conclusions: We identified a homeostatic circuit for the maintenance of iBAT metabolism that links the LAMTOR-mTORC1 pathway to PI3K-mTORC2-AKT signaling downstream of the insulin receptor., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

In this study, we assessed the efficacy of a novel Bacillus subtilis probiotic in improving growth performance and gut responses in comparison to pharmacological zinc oxide (ZnO) in nursery pigs. A total of 96 piglets were randomly assigned to four groups: Negative control (NC), Positive control (PC, 3000 mg Zn /kg feed), B.subtilis low dose (BS9-L, 2 × 10 7  CFU/pig) and B.subtilis high dose (BS9-H, 2 × 10 9  CFU/pig). Growth performance, diarrhea rate, gut mucosal gene expression and fecal microbial populations were evaluated. B.subtilis administration did not improve piglet bodyweight. BS9-L showed (P < 0.05) higher average daily gain (ADG) in Period 2 (D14-D28). BS9 groups had (P < 0.001) lower feed conversion ratio (FCR) in Period 2 (D14-D28) and overall. Like the ZnO-group, BS9 groups had lower (P < 0.01) diarrhea rate. A significant reduction (P < 0.05) in fecal E. coli, total coliforms, and an increase in lactic acid bacteria and Bacillus spp. in BS9 groups was observed. BS9 group had reduced (P < 0.05) mRNA levels of intestinal IL-8 and higher levels of MUC-1 and occludin and TJP-1 compared to negative control. These findings suggest that probiotic BS9, may promote growth performance, and ameliorate various indicators of intestinal health in piglets. Hence, it may serve as a prospective alternative to ZnO growth promoter in commercial swine production., (© 2023. The Author(s).)

Choline is an essential nutrient that is necessary for both fetal development and maintenance of neural function, while its effect on female ovarian development is largely unexplored. Our previous study demonstrated that choline supplementation promotes ovarian follicular development and ovulation, although its underlying mechanism was unclear. To uncover the potential regulation pathway, eighteen female Yorkshire × Landrace gilts were fed with either standard commercial diet (Control group, n = 9) or choline supplemented diet (Choline group, additional 500 mg/kg of control diet, n = 9) from day 90 of age to day 186. At day 186, feces samples were analyzed for effects on the gut microbiome using 16S ribosomal RNA gene V3-V4 region sequencing with Illumina MiSeq, serum samples were analyzed for trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) using HILIC method, and jejunum tissues were analyzed for immune related gene expression using qRT-PCR. Our results show that choline supplementation did not alter the circulating level of TMA and TMAO ( P > 0.05), but rather increased gut microbiome alpha diversity ( P < 0.05). Beta diversity analysis results showed that the choline diet mainly increased the abundance of Firmicutes , Proteobacteria , and Actinobacteria , but decreased the abundance of Bacteroidetes , Spirochaetes , and Euryarchaeota at the phyla level. Meta-genomic analysis revealed that choline supplementation activated pathways in the gut microbiota associated with steroid hormone biosynthesis and degradation of infertility-causing environmental pollutants (bisphenol, xylene, and dioxins). To further verify the effect of choline on intestinal activity, a porcine intestine cell line (IPEC-J2) was treated with serial concentrations of choline chloride in vitro . Our data demonstrated that choline promoted the proliferation of IPEC-J2 while inhibiting the apoptotic activity. qRT-PCR results showed that choline significantly increased the expression level of Bcl2 in both IPEC-J2 cells and jejunum tissues. The expression of IL-22 , a cytokine that has been shown to impact ovarian function, was increased by choline treatment in vitro . Our findings reveal the beneficial effect of choline supplementation on enhancing the gut microbiome composition and intestinal epithelial activity, and offer insights into how these changes may have contributed to the ovarian development-promoting effect we reported in our previous study., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhan, Fletcher, Huyben, Cai, Dingle, Qi, Huber, Wang and Li.)

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy 1 . The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) 2-5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN 6,7 . TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagC GDP -dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state., (© 2023. The Author(s).)

Epithelial polarization and polarized cargo transport are highly coordinated and interdependent processes. In our search for novel regulators of epithelial polarization and protein secretion, we used a genome-wide CRISPR/Cas9 screen and combined it with an assay based on fluorescence-activated cell sorting (FACS) to measure the secretion of the apical brush-border hydrolase dipeptidyl peptidase 4 (DPP4). In this way, we performed the first CRISPR screen to date in human polarized epithelial cells. Using high-resolution microscopy, we detected polarization defects and mislocalization of DPP4 to late endosomes/lysosomes after knockout of TM9SF4, anoctamin 8, and ARHGAP33, confirming the identification of novel factors for epithelial polarization and apical cargo secretion. Thus, we provide a powerful tool suitable for studying polarization and cargo secretion in epithelial cells. In addition, we provide a dataset that serves as a resource for the study of novel mechanisms for epithelial polarization and polarized transport and facilitates the investigation of novel congenital diseases associated with these processes., Competing Interests: KK, MH, ML, SH, KP, TM, GV, LH No competing interests declared, (© 2023, Klee, Hess et al.)

Standardized ileal digestibility (SID, %) of crude protein (CP) and amino acid (AA) and the metabolic availability (MA) of Met using the indicator amino acid oxidation (IAAO) method, in partially defatted black soldier fly larvae (PD-BSFL) meal were determined in growing pigs in 2 experiments. The Met SID value was then compared numerically with the Met MA to understand how different SID is compared with its MA value. In Exp. 1, 6 ileal-cannulated barrows (initial body weight [BW] = 18.03 ± 0.34 kg) were used in a 2-period switch back design and fed either a nitrogen-free diet (NFD) or test diet, with PD-BSFL meal as the sole source of AA, over two 11-d experimental periods, at a feeding level of 2.8 × estimated maintenance digestible energy requirement. Barrows were adapted for 9-d to the diet, followed by continuous 8-h ileal digesta collection on day 10 and 11. Digesta were pooled per pig within period. The SID of CP and Met of PD-BSFL meal were 76.1 ± 6.2% and 90.4 ± 3.9%, respectively. In Exp. 2, 7 barrows (initial BW = 18.77 ± 0.69 kg) were used in a 7 × 7 Latin square design with L-[1-13C]-Phe as the indicator AA. Each pig was randomly assigned to 1 of 7 dietary treatments over seven 3-d experimental periods. Two diet types were studied including reference (crystalline AA) and PD-BSFL test diets, each supplying graded intakes of Met at 55, 65, and 75% of the estimated SID requirement (NRC, 2012). The MA of Met was determined by comparing the IAAO response between the reference and PD-BSFL test diet using the slope-ratio method. Linear regression determined a negative slope of the best fit line for both the reference and test diets (P < 0.05). The MA of Met in PD-BSFL meal was 53.3%, which is as expected lower than the SID value. While it is generally appreciated that MA will be less than SID, the use of SID is more practical. In cases where SID cannot explain physiological outcomes of feeding a novel ingredient, IAAO may provide additional insight into whether MA should be explored., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Eighty-eight gilts [initial body weight (BW) 49.8 ± 0.8 kg] were recruited to determine the effects of moderate energy and protein restriction during the development period on changes in BW and backfat depth (BF) and subsequent lactation performance. Gilts were randomly assigned to one of four feeding programs: 1) standard commercial diet fed ad libitum (CON), 2) standard commercial diet fed 10% or 3) 20% below ad libitum, or 4) a high-fiber diet fed ad libitum [2.5 times more fiber (neutral detergent fiber) than the commercial diet to dilute net energy and crude protein by approximately 20% and 13%, respectively; FIB]. The gilts were housed individually and received the feeding programs between 90 and 190 (breeding) d of age and standard gestation and lactation diets thereafter. Litters were standardized to 12 ± 1 pigs within 48 h of farrowing; weaning occurred at 20.0 ± 0.4 d of age. Gilts that received the 20% restricted program had lower overall average daily feed intake (ADFI) during the development period (2.64 ± 0.04 kg; P < 0.05) versus all other feeding programs and gilts that received the 10% restricted program had lower ADFI than FIB and CON, which were not different (2.96 vs. 3.44 and 3.47 ± 0.04 kg for 10%, FIB and CON, respectively; P < 0.05). Plasma free fatty acid (FFA) concentrations on day 180 of age were lower for gilts that received the 10% and 20% restricted programs compared to gilts that received the FIB and CON programs, which were not different (97 and 86 vs. 220 and 149 ± 29 µEq/L, respectively; P < 0.05). Plasma concentrations of glucose, urea, prolactin, and IGF-1 were not different among feeding programs on day 180 of age. At breeding, gilts that received the FIB and 10% programs had lower BW and BF versus CON (145.7 and 144.8 vs. 155.2 ± 0.9 kg and 14.4 and 14.8 vs. 16.5 ± 0.2 mm for BW and BF, respectively; P < 0.05) but greater BW than gilts that received the 20% restricted program (137.9 kg; P < 0.05). The BW and BF of gilts did not differ at the end of gestation or at weaning. The ADFI of sows during lactation and offspring birth weight and growth rate during lactation and the 5-wk nursery period were not influenced by gilt development feeding program. Therefore, a high-fiber feeding program could be used in group-housing gilt-development scenarios, where feed is offered ad libitum, to control BW and BF prior to breeding without influencing milk production in the subsequent lactation period., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This project was conducted to determine if providing standardized ileal digestible (SID) Lys at 40% above estimated requirements (NRC, 2012), with the concomitant increased protein intake, from days 90 to 110 of gestation stimulates mammary development in multiparous sows. From day 90 of gestation, Yorkshire × Landrace multiparous sows (parities 2 and 3) were fed 2.6 kg/d of either a conventional diet (CTL, control, n = 17) providing 14.8 g/d of SID Lys or a diet providing 20.8 g/d of SID Lys via additional soybean meal (HILYS, n = 16). The diets were isoenergetic. Concentrations of IGF-1, glucose, free fatty acids (FFA), urea, and amino acids (AA) were measured in jugular blood samples obtained on days 90 and 110 of gestation. Sows were necropsied on day 110 ± 1 of gestation to obtain mammary glands for compositional and histological analyses. Backfat or BW changes of sows during late gestation were unaffected by treatment (P > 0.10), as was the case for fetal BW (P > 0.10). None of the variables measured in mammary tissue were altered by supplementary Lys (P > 0.10). Circulating IGF-1, glucose, and FFA did not differ (P > 0.10) between HILYS and CTL sows on day 110 of gestation, whereas concentrations of urea were greater (P < 0.01) in HILYS versus CTL gilts. Concentrations of Ile and Thr in plasma were also greater (P < 0.05), and those of Glu were lower (P < 0.01) in HILYS than CTL sows. These results demonstrate that feeding Lys (via protein) above current NRC recommendations during late gestation does not improve mammary development of multiparous sows. Hence, the use of a two-phase feeding strategy to provide more Lys (protein) to multiparous sows during this period is not necessary., (© His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2023.)

Here, we describe a detailed step-by-step protocol for the detection of phosphoproteins in two-dimensional difference gel electrophoresis (2D-DIGE) gels. A standard 2D-DIGE protocol is combined with subsequent post-staining with phosphospecific fluorescent dye. The combination of these two methods complements 2D-DIGE-based proteome profiling by fluorescence detection of phosphoproteins in the same gel providing additional possibility for sensitive and accurate quantification of the differentially regulated phosphoproteins in biological samples., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Black soldier fly larvae meal (BSFLM) is a relatively new ingredient that has attracted attention for application in human, farm, and companion animal nutrition. However, there is limited data on protein quality (PQ) of BSFLM. This study compared PQ in BSFLM with soybean meal (SBM) and fishmeal (FM) using the classical protein efficiency ratio (PER) chick growth assay model. A total of 240-male day-old Ross × Ross broiler chicks were allocated to 6 test diets for 10 d test. The corn starch-dextrose based diets were: 5 test diets containing 10% crude protein (CP) from casein (as a standard assay diet), SBM, FM, or BSFLM without or with (BSFLM+) additional essential amino acids (EAA) to match the levels of the SBM diet on digestible basis. A sixth diet was nitrogen-free (NFD), to enable calculation of net protein ratio (NPR). The birds had ad libitum access to feed and water, body weight (BW) and feed intake (FI) were recorded at the beginning and end of the experimental feeding period. Body weight gain (BWG), gain to feed ratio (G:F), crude protein intake (CPI), PER, and NPR were calculated. The analyzed CP (as fed) was 10.1, 12.8, 9.5, 11.7, and 14.9% for casein, SBM, FM, BSFLM, and BSFLM+ diets, respectively. Birds fed BSFLM+ had greater BWG (P < 0.01) than birds fed other diets. Feed intake was greater (P < 0.01) for birds fed BSFLM+ and FM diets than birds fed other diets. Birds fed BSFLM+ ate (P < 0.01) more CP than birds fed other diets. Diets had no (P > 0.05) effects on PER and NPR. In terms of ranking, the PER was 2.44, 2.38, 2.34, 2.28, and 2.10 g BWG/g CPI for BSFLM+, SBM, FM, casein, and BSLM, respectively. Corresponding values for NPR were 2.69, 2.78, 2.70, 2.74, and 2.48 g BWG/g CPI, respectively. In conclusion, the protein quality of BSFLM without or with additional EAA was comparable with FM and SBM., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Black soldier fly larvae meal (BSFLM) is characterized with good nutritional and functional attributes. However, there is limited data on inclusion of BSFLM in broiler chicken rations from placement through to market weight. Therefore, we examined growth and organ responses of partial to complete replacement of soybean meal (SBM) with BSFLM in a practical feeding program. A total of 1,152 d-old male Ross × Ross 708 chicks were allocated to 48 pens and assigned one of six diets (n = 8). The diets were: a basal corn-SBM diet (0%BSFLM), 4 diets in which SBM in 0%BSFLM was replaced with BSFLM at 12.5, 25, 50, and 100% and a final diet (0 + AGP) in which 0%BSFLM was treated with coccidiostat (70 mg Narasin/kg) and antibiotic (55 mg Bacitracin Methylene Disalicylate/kg). For energy fortification, soy oil was used for 0%BSFLM diets and black soldier fly oil in the other diets. Body weight, feed intake (FI), BW gain (BWG), and mortality-corrected feed conversion ratio (FCR) were reported. Organ weights were recorded on d 24 and 49. On d 10, birds fed diets 12.5, 25, and 0 + AGP had higher BWG than birds fed diets 0, 50, and 100 (P < 0.01), and birds fed diet 100 had lower BWG than birds fed diets 0 or 50 (P < 0.01). Birds fed diets 50 and 100 had lower BWG than birds fed all other diets on d 24 and 49 (P < 0.05). Overall (d 0-49), BSFLM linearly (P < 0.01) decreased BW, BWG, and FI and increased FCR and mortality. The overall BWG of 50 and 100% BSFLM birds was 92 and 81% of birds fed 0%BSFLM, repectively and coresponding overall FI was 96 and 90%. An increase in gizzard, small intestine, pancreas, and liver relative weights were observed with increasing BSFLM inclusion (P < 0.01). The data indicated that lower levels of BSFLM could provide some growth-promoting effects commensurate to antibiotics in the starter phase. However, replacing SBM with greater amounts (≥50) of BSFLM reduced growth and increased organ size., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The selection of sows that are reproductively fit and produce large litters of piglets is imperative for success in the pork industry. Currently, low heritability of reproductive and litter-related traits and unfavourable genetic correlations are slowing the improvement of pig selection efficiency. The integration of biomarkers as a supplement or alternative to the use of genetic markers may permit the optimization and increase of selection protocol efficiency. Metabolite biomarkers are an advantageous class of biomarkers that can facilitate the identification of cellular processes implicated in reproductive condition. Metabolism and metabolic biomarkers have been previously implicated in studies of female mammalian fertility, however a systematic analysis across multiple biofluids in infertile and high reproductive potential phenotypes has not been explored. In the current study, the serum, urinary and salivary metabolomes of infertile (INF) sows and high reproductive potential (HRP) sows with a live litter size ≥ 13 piglets were examined using LC-MS/MS techniques, and a data pipeline was used to highlight possible metabolite reproductive biomarkers discriminating the reproductive groups. The metabolomes of HRP and INF sows were distinct, including significant alterations in amino acid, fatty acid, membrane lipid and steroid hormone metabolism. Carnitines and fatty acid related metabolites were most discriminatory in separating and classifying the HRP and INF sows based on their biofluid metabolome. It appears that urine is a superior biofluid than saliva and serum for potentially predicting the reproductive potential level of a given female pig based on the performance of the resultant biomarker models. This study lays the groundwork for improving gilt and sow selection protocols using metabolomics as a tool for the prediction of reproductive potential.

Six ileal-cannulated barrows (28.0 ± 1.3 kg initial BW) were used in a replicated 3 × 3 Latin square design with one additional period ( n = 7 or 6) to determine standardized ileal digestible (SID) AA and digestible energy of two modified soy protein concentrates [MSPC1 and MSPC2] and soybean meal (SBM). Pigs were fed one of three cornstarch-based diets with either MSPC1 or MSPC2 or SBM as the sole source of AA at a rate of 2.8 times the estimated maintenance energy requirement. In each period, pigs were adapted to diets for 7 d followed by 2 d of fecal collection and subsequently, 2 d of continuous ileal digesta collection for 8 h. The SID of AA was calculated using basal endogenous losses from a previous study for pigs fed a nitrogen-free diet. The digestible energy of the ingredients was calculated according to the difference method using a nitrogen-free diet that contained the same cornstarch:sucrose:oil ratio as the three test diets. The total Lys content was 33% and 38% greater for MSPC1 vs. MSPC2 and SBM, respectively. The SID of crude protein was greater for MSPC1 (96.9%) than for SBM (91.3%; P < 0.05), whereas an intermediate value was observed for MSPC2 (94.3% ± 1.2%). The SID of Ile (93.8%), Leu (93.6%), Lys (93.9%), Phe (96.7%), and Val (93.2%) were not different between MSPC1 and MSPC2 but greater than for SBM (88.8% ± 1.3%, 87.8% ± 1.2%, 84.5% ± 1.7%, 92.9% ± 1.0%, 86.5% ± 1.7% for Ile, Leu, Lys, Phe, and Val, respectively; P < 0.05). The SID of His and Thr was greater for MSPC1 than MSPC2 and SBM ( P < 0.05), which were not different. The SID of Met was greater for MSPC1 and SBM vs. MSPC2 ( P < 0.05). The SID of Arg was greater for MSPC1 than MSPC2 and SBM ( P < 0.05), and greater for MSPC2 than SBM ( P < 0.05). The digestible energy was greater for MSPC1 (4,677 kcal/kg) than MSPC2 and SBM (average; 3,896 ± 239 kcal/kg; P < 0.05), which were not different. Therefore, the MSPC1 was a better source of SID Lys and digestible energy than either MSPC2 or SBM and could be used as a highly digestible protein ingredient in swine rations., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Three hundred twenty newly weaned pigs (21 days of age; 6.7 ± 0.3 kg BW) were used to determine the effects of supplementing low-complexity ( LC ) deoxynivalenol- ( DON ) contaminated nursery diets with a feed additive or fish oil on growth performance and immune response to an Escherichia coli lipopolysaccharide ( LPS ) challenge. Pens were randomly assigned to 1 of 5 dietary treatments ( n = 8 pens per treatment): positive control ( PC ; contained multiple animal protein sources), or 1 of 4 LC diets (contained only plant-based protein sources) without ( NC ; negative control) or with ~ 3.5 ppm DON contamination, without ( DON- ) or with a feed additive containing a blend of immune-modulating components ( DON+ ; 2 mg/kg, as-fed) or fish oil ( DONω3 ; 2.5%, as-fed). Dietary treatments were fed during phases I and II (7 and 15 days, respectively) and a common phase III diet was fed for 20 days. On day 22, two pigs per pen were injected IM with 30 μg/kg BW LPS and 1 pig per pen with 1 mL saline. Rectal temperatures were recorded at 0, 1, 2, 3 h after injection. At 3 h, blood was collected for plasma cytokine analysis and small intestinal histomorphology was assessed. In phase I, pigs fed PC and NC did not differ for ADG, ADFI and G:F, but these outcomes were greater than for pigs fed DON+ and DONω ( P < 0.05). In phase II, pigs fed NC had greater ADG and PC had greater ADFI but lower G:F than pigs fed DON- and DONω3 ( P < 0.05). At the end of phase II, pigs fed DONω3 tended to have lower BW than PC and NC ( P = 0.084 and 0.079, respectively). In phase III and overall, there were no differences among dietary treatments for ADG, ADFI, G:F, or final BW. The LPS injection increased rectal temperature and reduced jejunal and ileal villus height (versus saline; P < 0.05). Plasma interferon-γ concentration was only increased by LPS for pigs fed PC, NC, and DON+ compared to the saline-injected counterparts ( P < 0.05). Regardless of LPS injection, jejunal villus height was greater for pigs fed DON+ than DONω3 ( P < 0.05) and ileal villus height was greater for pigs fed DON+ and PC than DONω3 ( P < 0.05). Therefore, nursery diet complexity did not affect growth performance or immune response to LPS. Regardless of DON contamination and feed additive inclusion in phases I and II, pigs were able to achieve nursery exit BW not different from those fed PC. The feed additive offered marginal benefits for small intestinal villus height and immune response for pigs fed DON-contaminated LC nursery diets., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Fifty-six litters from first-parity sows standardized to 12 piglets were used to determine the effects of creep feed composition and form and the provision of low- or high-complexity nursery diets on the evolution of small intestinal histomorphology and jejunal mucosa-specific enzyme activities postweaning. At 5 d of age, litters (initial bodyweight [BW] 2.31 ± 0.61 kg) were assigned to one of four creep feeding regimens (n = 14): 1) commercial creep feed (COM), 2) liquid milk replacer (LMR), 3) pelleted milk replacer (PMR), or 4) no creep feed (NO). At weaning (21 d of age), six pigs per litter were provided a HIGH- (contained highly digestible animal proteins) or LOW- (contained corn and soybean meal as main protein sources) complexity nursery diet (n = 7). At 21, 28, and 59 d of age, two pigs per pen (one castrated male and one female) were euthanized, and ileal and jejunal segments for histomorphological measurements and jejunal mucosal scrapings were collected to determine specific mucosa enzyme activities. At weaning, pigs provided COM had a greater ileal absorptive capacity (M) than LMR or NO, which were not different (14.1 vs. 10.4 and 10.5 ± 0.9 μm2; P < 0.05); PMR was intermediate. On days 28 and 59, M was not different among pigs regardless of creep feed treatments. Pigs fed LOW had reduced jejunal villus height (VH; P < 0.001) and M (P < 0.001) on day 28 vs. day 21. The VH and M were not different for pigs fed HIGH or LOW by the end of the nursery period. For all dietary treatments except COM-HIGH and COM-LOW, jejunal mucosal maltase-specific activity was not different between days 21 and 28 of age but greater on day 59 (P < 0.05). For pigs that received COM-HIGH, maltase-specific activity was not different between days 21 and 28 but greater on day 59 than day 28 (P < 0.05). For pigs that received COM-LOW, maltase-specific activity was not different between days 21, 28, and 59. Regardless of creep or nursery treatment, sucrase-specific activity was the greatest on day 59, followed by days 21 and 28 (P < 0.001), and lactase-specific activity was greater on day 21 than on days 28 and 59 (P < 0.001), which were not different. Therefore, pigs that provided LOW diet had greater villus atrophy and reduced M during the first week after weaning vs. pigs that provided HIGH, regardless of creep feeding regimen, but were able to recover by the end of the nursery period., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

The goal of this project was to determine if standardized ileal digestible (SID) lysine provided at 40% above estimated requirements, with the concomitant increase in protein intake, from days 90 to 110 of gestation would stimulate mammary development in gilts. From day 90 of gestation, Yorkshire × Landrace gilts were fed 2.65 kg of either a conventional diet (CTL, control, n = 19) providing 18.6 g/d of SID Lys or a diet providing 26.0 g/d of SID Lys via additional soybean meal (HILYS, n = 19). Both diets were isoenergetic. Jugular blood samples obtained on days 90 and 110 of gestation were used to measure concentrations of insulin-like growth factor-1 (IGF-1), metabolites, and amino acids (AA). Gilts were necropsied on day 110 ± 1 of gestation to obtain mammary glands for compositional analyses, immunohistochemistry, and analysis of mRNA abundance for AA transporters and markers of cell proliferation and differentiation. The HILYS gilts gained more body weight (P < 0.01) during the experimental period compared with CTL gilts, and had greater fetal weights (1.29 vs. 1.21 ± 0.03 kg, P < 0.05). There was no difference in circulating IGF-1, glucose, or albumin (P > 0.10) between HILYS and CTL gilts on day 110 of gestation, whereas concentrations of urea and free fatty acids were greater (P < 0.01), and those of Trp and Ala were lower (P < 0.05), in HILYS than CTL gilts. The provision of lysine at 40% above estimated requirements increased total mammary parenchymal mass by 44%, as well as total parenchymal fat, protein, DNA, and RNA (P < 0.01). The mRNA abundance of ACACA was greater (P < 0.05) in HILYS than CTL gilts, while only the AA transporter SLC6A14 tended (P < 0.10) to be greater. Results demonstrate that providing dietary Lys above current National Research Council recommendations in late gestation increases mammary development in gilts. Results also indicate that Lys may have been limiting for protein retention. These data suggest that the use of a two-phase feeding strategy during gestation, whereby dietary Lys is increased from day 90, could benefit potential sow milk yield in the subsequent lactation., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food, Government of Canada, 2022.)

Objectives: Metabolic inflammation is a hallmark of obesity and related disorders, afflicting substantial morbidity and mortality to individuals worldwide. White visceral and subcutaneous adipose tissue not only serves as energy storage but also controls metabolism. Adipose tissue inflammation, commonly observed in human obesity, is considered a critical driver of metabolic perturbation while molecular hubs are poorly explored. Metabolic stress evoked by e.g. long-chain fatty acids leads to oxidative perturbation of adipocytes and production of inflammatory cytokines, fuelling macrophage infiltration and systemic low-grade inflammation. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation, accumulation of oxygen-specific epitopes and cell death, collectively referred to as ferroptosis. Here, we explore the function of adipocyte GPX4 in mammalian metabolism., Methods: We studied the regulation and function of GPX4 in differentiated mouse adipocytes derived from 3T3-L1 fibroblasts. We generated two conditional adipocyte-specific Gpx4 knockout mice by crossing Gpx4 fl/fl mice with Adipoq-Cre + (Gpx4 -/-AT ) or Fabp4-Cre + (Gpx4 +/-Fabp4 ) mice. Both models were metabolically characterized by a glucose tolerance test and insulin resistance test, and adipose tissue lipid peroxidation, inflammation and cell death were assessed by quantifying oxygen-specific epitopes, transcriptional analysis of chemokines, quantification of F4/80 + macrophages and TUNEL labelling., Results: GPX4 expression was induced during and required for adipocyte differentiation. In mature adipocytes, impaired GPX4 activity spontaneously evoked lipid peroxidation and expression of inflammatory cytokines such as TNF-α, interleukin 1β (IL-1β), IL-6 and the IL-8 homologue CXCL1. Gpx4 -/-AT mice spontaneously displayed adipocyte hypertrophy on a chow diet, which was paralleled by the accumulation of oxygen-specific epitopes and macrophage infiltration in adipose tissue. Furthermore, Gpx4 -/-AT mice spontaneously developed glucose intolerance, hepatic insulin resistance and systemic low-grade inflammation, when compared to wildtype littermates, which was similarly recapitulated in Gpx4 +/-Fabp4 mice. Gpx4 -/-AT mice exhibited no signs of adipocyte death., Conclusion: Adipocyte GPX4 protects against spontaneous metabolic dysregulation and systemic low-grade inflammation independent from ferroptosis, which could be therapeutically exploited in the future., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Inflammatory skin diseases, including atopic dermatitis (AD) and psoriasis, are increasing in populations worldwide. The treatment of patients with AD and other forms of skin inflammation is mainly based on the use of topical corticosteroids or calcineurin inhibitors, which can cause significant side effects with long-term use. Therefore, there is a great need for the development of more effective and less toxic anti-inflammatory agents suitable for the treatment of chronic skin lesions. Here, we screened a number of strains from the ASIB 505 terrestrial algae collection and identified a green algae Chromochloris zofingiensis with pronounced anti-inflammatory properties. We found that a crude nonpolar extract of C. zofingiensis (ID name NAE&#95;2022C), grown upon nitrogen deprivation, acts as a bioactive substance by inhibiting TNFR/NF-κB responses in human skin keratinocyte HaCaT cells. We also found that NAE&#95;2022C suppressed the secretion of pro-inflammatory cytokine tumor necrosis factor α (TNFα) and several Th1- and Th2-related chemokines in a reconstituted human epidermis. The TNFR/NF-κB pathway analysis showed multiple inhibitory effects at different levels and disclosed a direct targeting of IKKβ by the extract. Bioassay-guided fractionation followed by high-resolution mass spectrometry detected diacylglyceryl-trimethylhomoserine (DGTS), Lyso-DGTS (LDGTS), 5-phenylvaleric acid, theophylline and oleamide as leading metabolites in the active fraction of NAE&#95;2022C. Further analysis identified betaine lipid DGTS (32:0) as one of the active compounds responsible for the NAE&#95;2022C-mediated NF-κB suppression. Overall, this study presents an approach for the isolation, screening, and identification of anti-inflammatory secondary metabolites produced by soil algae.

Growth performance and physiological responses of nursery piglets when fed enzymatically treated yeast (HY40) and pharmacological ZnO alone or in combination were investigated. A total of 144 pigs (21 d old, BW 7.32 ± 0.55 kg) were placed in 36 pens (4 pigs/pen). Pigs were randomly assigned to one of four dietary treatments (n = 9): 1) control corn-wheat-soybean meal diet (control), 2) control + HY40 (HY40), 3) control + (ZnO) and 4) control + HY40 + ZnO (HY40+ZnO). Inclusion of HY40 and ZnO was 0.5% and 3,000 ppm in phase I (days 0 to 14), respectively, and halved in phase II (days 15 to 42). All diets contained 0.2% TiO2 for determination of apparent total tract digestibility (ATTD) of components. Body weight and feed disappearance were recorded weekly. One pig per pen was killed for organ weights, blood, and tissue samples on day 14. Except for phase II, when HY40 + ZnO pigs had greater average daily feed intake (P = 0.004) than all other treatments, there were no (P > 0.05) interactions between HY40 and ZnO on growth performance. Pigs fed HY40 or ZnO containing diets were heavier (P < 0.05) than pigs fed without by the end of the study. On day 14, pigs fed additives exhibited higher (P ≤ 0.009) ATTD of dry matter (DM) and gross energy (GE) than control pigs. On day 28, pigs fed control, HY40, and HY40 + ZnO had greater (P ≤ 0.022) ATTD of DM, crude protein, and GE than piglets fed ZnO only. Pigs fed HY40 + ZnO had lower ileal digesta Escherichia coli concentration (P < 0.05) than HY40 and control pigs. Ileal digesta of pigs fed ZnO diets had higher lactobacillus to E. coli ratio (1.44 vs. 1.20; P = 0.001), exhibited higher concentrations of acetic (P = 0.01) and butyric acid (P = 0.01) but lower lactic (P = 0.02) and total short chain fatty acids (P = 0.033) than pigs fed non-ZnO diets. Greater (P < 0.05) mRNA expression of nutrient transporters, tight junction proteins, and fecal excretion of zinc (Zn) was observed in ZnO pigs relative to non-ZnO pigs. Pigs fed HY40 diets had greater (P = 0.002) villus height to crypt depth ratio (VH:CD) than non-HY40 pigs. The concentration of plasma IgA was higher (P = 0.04) in HY40 + ZnO pigs relative to other pigs, whereas HY40 pigs showed higher (P < 0.001) jejunal IgA than non-HY40 pigs. Although the mode of action of HY40 and ZnO differed, the present study indicated that HY40 improved growth performance and jejunal function and immunity, making HY40 an effective alternative to pharmacological ZnO in nursery pigs feeding programs., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Background: Female infertility is a health issue for both humans and animals and despite developments in medical interventions, there are still some conditions that cannot be treated successfully. It is important to explore other potential therapies or remedies that could improve reproductive health. Choline is an over-the-counter supplement and essential nutrient that has many health benefits. It has been suggested to be beneficial in various aspects of fertility, including fetal development and endocrine disorders like polycystic ovarian syndrome (PCOS). However, choline's impact on ovarian function has not been explored., Methods: To study the effects of choline on ovarian development, 36 female Yorkshire × Landrace pigs were fed the following four supplemented diets between 90 and 186 days of age: (1) Control (corn and soybean meal-based diet that met estimated nutrient requirements, n = 9); (2) Choline (additional 500 mg choline per 1 kg of control diet, n = 8); (3) Omega-3 (additional 5556 mg Omega-3 per 1 kg control diet by introducing fish oil); (4) Choline + Omega-3 (500 mg choline + 5556 mg Omega-3 per 1 kg control diet). Pigs fed the choline-supplemented diet were compared to the control group and those fed diets supplemented with Omega-3 as fertility-promoting agent., Results: It was found that the number of corpus luteum per ovary in the Choline (16.25 ± 2.88), Omega-3 (10.78 ± 1.71) and Choline + Omega-3 (14.89 ± 2.97) groups were all higher in comparison to that of the control group (5.56 ± 1.72, p < 0.05). The percentage of antral follicles in the Choline + Omega-3 group were higher compared to the control group (p < 0.05). To elucidate the potential molecular mechanism of choline on these improved ovarian phenotypes, the expression of a group of genes that are involved in ovarian development, including cytochrome P450 family 11 subfamily A member 1 (CYP11A1), follicle stimulating hormone receptor (FHSR) and luteinizing hormone receptor (LHR), was analyzed using RT-qPCR. The expression of both LHR and CYP11A1 was significantly upregulated in the choline-supplemented group (p < 0.05), while there are no differences in FSHR expression among all the groups. Additionally, the expression of miR-21, -378, -574, previously found to be important in ovarian function, were examined. Our data showed that miR-574 was upregulated in the Choline group while miR-378 was upregulated in the Choline + Omega-3 group in comparison to the control group (p < 0.05). Further, serum metabolite analysis showed that 1-(5Z, 8Z, 11Z, 14Z, 17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, a form of phosphatidylcholine metabolite, was significantly increased in all the treatment groups (p < 0.05), while testosterone was significantly increased in both Omega-3 and Choline + Omega-3 groups (p < 0.05) and tended to be reduced in the choline-supplemented group (p = 0.08) compared to the control group., Conclusions: Our study demonstrated choline's influence on ovarian function in vivo, and offered insights into the mechanisms behind its positive effect on ovarian development phenotype., (© 2021 The Author(s). Published by BRI.)

The objective of the current study was to determine the effects of precisely meeting estimated daily energy and Lys requirements for gestating sows over three consecutive pregnancies on sow reproductive and lactation performance. A total of 105 sows (initial reproductive cycle 1.4 ± 0.5) were randomly assigned to a precision (PF; n = 50) or control (CON; n = 55) feeding program between days 2 and 9 of gestation and housed in group-pens equipped with electronic sow feeders capable of blending two diets. The PF sows received unique daily blends of two isocaloric diets (2518 kcal/kg NE; 0.80% and 0.20% standardized ileal digestible [SID] Lys, respectively), whereas CON sows received a static blend throughout gestation to achieve 0.56% SID Lys. After weaning, sows were re-bred and entered the same feeding program as in the previous pregnancy for two subsequent pregnancy cycles (PF: n = 36; CON: n = 37; average reproductive cycle: 2.4 ± 0.5; PF: n = 25; CON: n = 24; average reproductive cycle: 3.5 ± 0.5). Sows on the PF program received 97%, 105%, and 118% (average over three pregnancy cycles) of dietary energy and 67%, 79%, and 106% of SID Lys intakes compared to CON between days 5 and 37, 38 and 72, and 73 and 108 of gestation, respectively. Estimated N (26.1%) retention did not differ between gestation feeding programs in any pregnancy, but excess N excretion was less (1617 vs. 1750 ± 54 g/sow; P < 0.01) for PF vs. CON sows. Regardless of pregnancy cycle, sows that received the PF program had greater ADG between days 38 and 72 (614 vs. 518 ± 63 g/d; P < 0.05) and between days 73 and 108 (719 vs. 618 ± 94 g/d; P = 0.063) of gestation, and greater loin depth gain between days 63 and 110 of gestation (0.7 vs. -1.1 ± 1.6 mm; P < 0.05), but BW (235.1 kg) and backfat (17.8 mm) and loin (70.5 mm) depths on day 110 of gestation did not differ. The number of piglets born alive, stillborn, and mummified, and litter birth weight (16.5 kg) did not differ in any pregnancy cycle, nor did piglet ADG during lactation (250 g/d) and piglet BW (6.7 kg) at weaning. Sows that received the PF program during gestation had lower ADFI during lactation (5.7 vs. 6.2 ± 0.2 kg; P < 0.01). Therefore, using feeding programs that precisely match estimated daily energy and Lys requirements for gestating sows provides the opportunity to reduce N losses to the environment and reduce lactation feed usage, without negatively affecting sow reproductive and lactation performance., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science.)

A total of 601 pigs from 65 litters were used to determine the effects of closely meeting estimated daily Lys and energy requirements for sows during gestation for three consecutive parities on offspring postweaning growth performance and carcass and loin quality at slaughter. Sows were assigned a control (static diet composition; CON ) or precision (individual daily blend of two diets to meet estimated Lys and energy requirements; PRE ) feeding program between days 7 and 110 of gestation for three consecutive pregnancy cycles, starting with primiparous sows (parity 1: 12 CON and 12 PRE sows; parity 2: 8 CON and 13 PRE sows; parity 3: 8 CON and 12 PRE sows). At weaning (20 ± 2 d of age), up to 10 pigs per litter were randomly selected and placed in a pen (1 litter per pen). All pens received ad libitum access to commercial diets in six phases (four-phase nursery, grower, and finisher, respectively). Four pigs per pen were slaughtered at ~125 kg BW for evaluation of carcass characteristics and loin quality. The ADG and ADFI of offspring were not influenced by maternal feeding program in any parity during nursery phases I through III. During nursery phase IV, ADG and ADFI were greater for litters from PRE- vs. CON-fed sows (0.70 vs. 0.66 ± 0.03 and 1.15 vs. 1.08 ± 0.06 kg/d for ADG and ADFI, respectively; P < 0.05). The BW for litters from PRE- vs. CON-fed sows tended to be greater by day 66 of age (end of nursery period; 29.7 vs. 28.7 ± 1.1 kg; P = 0.076). Within the grower phase, litters from PRE-fed sows had a greater ADG in parity 2 but lower ADG in parity 3 vs. litters from CON-fed sows (0.99 vs. 0.94 and 0.93 vs. 1.01 ± 0.03 kg/d for parities 2 and 3, respectively; P < 0.05). No differences were observed for ADG or ADFI in the finisher phase or G:F in any phase for any parity. Loin eye area was smaller (52.2 vs. 55.0 ± 1.8 cm 2 ; P < 0.05) for offspring from PRE- vs. CON-fed sows. In parity 2, carcass lean yield tended to be less for offspring from PRE- vs. CON-fed sows (58.6 vs. 59.6 ± 0.4%; P = 0.051). Minimal differences were observed for subjective and objective evaluations of loin quality. Closely meeting the estimated daily energy and Lys requirements for sows throughout gestation for three consecutive pregnancy cycles improved offspring growth performance (ADG and ADFI) in the final nursery stage, but generally did not affect growth performance in grower/finisher periods or carcass and loin quality at ~125 kg BW., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Fifty-six litters from first-parity sows standardized to 12 piglets were used to determine the effects of creep feed composition and form on pre- and post-weaning pig growth performance and the utilization of low-complexity nursery diets. At 5 days of age, litters (initial body weight [BW] 2.31 ± 0.61 kg) were assigned to one of four creep feeding regimens ( n = 14): 1) pelleted commercial creep feed (COM), 2) liquid milk replacer (LMR), 3) pelleted milk replacer (PMR), or 4) no creep feed (NO); creep feeds contained 1.0% brilliant blue as a fecal marker. Individual piglet BW and fecal swabs were collected every 3 ± 1 days during the creep-feeding period. The latter was to identify piglets that regularly consumed creep feed via the visual appearance of blue dye in the feces. At weaning (21 ± 2 days of age), six pigs per litter with median BW that consumed creep feed were placed on either a HIGH- (contained highly digestible animal proteins) or LOW- (contained corn and soybean meal as the main protein sources) complexity nursery diet ( n = 7) in a three-phase feeding program over 39 days. On day 8, two pigs per pen were sacrificed to collect organ weights and digesta. The LMR disappeared at the greatest rate (average 37.7 g/pig/d; dry matter-basis) versus COM and PMR (10.6 and 10.3 ± 1.5 g/pig/d, respectively; P < 0.001). Litters that received LMR had the greatest proportion of pigs with blue fecal swabs throughout the creep feeding period (85.0 vs. 54.9 and 63.0% ± 0.4% for COM and PMR, respectively; P < 0.05) and LMR piglets had greater BW at weaning versus all other treatments (6.32 vs. 6.02, 5.92, and 5.67 ± 0.14 kg, for LMR, COM, NO, and PMR, respectively; P < 0.001). Overall, pigs given LOW (vs. HIGH) diets in the nursery period had reduced average daily gain (25.1 vs. 27.7 ± 0.4 g/kg BW; P < 0.001), gain:feed (0.75 vs. 0.81 ± 0.02; P < 0.001), and exit BW (21.2 vs. 24.4 ± 0.6 kg; P < 0.001); no carryover effects of creep feeding program were observed. Creep feed regimen had limited effects on nutrient digestibility of nursery diets but the apparent ileal digestibility of organic matter tended to be less at 28 days of age for pigs that received the LOW nursery diet (64.2 vs. 68.8% ± 2.5%; P = 0.076). Providing supplemental nutrition during the suckling period via LMR improved piglet BW at weaning, which did not correspond to improved post-weaning growth performance, regardless of nursery diet complexity., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Complement-opsonized HIV-1 triggers efficient antiviral type I interferon (IFN) responses in dendritic cells (DCs), which play an important role in protective responses at the earliest stages in retroviral infection. In contrast, HIV-1 suppresses or escapes sensing by STING- and MAVS-associated sensors. Here, we identified a complement receptor-mediated sensing pathway, where DCs are activated in CCR5/RLR (RIG-I/MDA5)/MAVS/TBK1-dependent fashion. Increased fusion of complement-opsonized HIV-1 via complement receptor 4 and CCR5 leads to increased incoming HIV-1 RNA in the cytoplasm, sensed by a nonredundant cooperative effect of RIG-I and MDA5. Moreover, complement-opsonized HIV-1 down-modulated the MAVS-suppressive Raf-1/PLK1 pathway, thereby opening the antiviral recognition pathway via MAVS. This in turn was followed by MAVS aggregation and subsequent TBK1/IRF3/NF-κB activation in DCs exposed to complement- but not non-opsonized HIV-1. Our data strongly suggest that complement is important in the induction of efficient antiviral immune responses by preventing HIV-1 suppressive mechanisms as well as inducing specific cytosolic sensors. IMPORTANCE Importantly, our study highlights an unusual target on DCs-the α chain of complement receptor 4 (CR4) (CD11c)-for therapeutic interventions in HIV-1 treatment. Targeting CD11c on DCs mediated a potent antiviral immune response via clustering of CR4 and CCR5 and subsequent opening of an antiviral recognition pathway in DCs via MAVS. This novel finding might provide novel tools for specifically boosting endogenous antiviral immunity via CR4, abundantly expressed on multiple DC subsets.

Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.