High Protein Research Articles

Water deprivation induces a systemic pro-catabolic state that differentially affects oxidative and glycolytic skeletal muscles in male mice.

Dehydration, characterized by the loss of total body water and/or electrolytes due to diseases or inadequate fluid intake, is prevalent globally but often underestimated. Its contribution to long-term chronic diseases and sarcopenia is recognized, yet the mechanisms involved in systemic and muscle protein metabolism during dehydration remain unclear. This study investigated metabolic adaptations in a 36-hour water deprivation (WD) model of mice. Male C57BL/6 mice underwent 36-h WD or pair-feeding at rest, with assessments of motor skills along with biochemical, and metabolic parameters. Dehydration was confirmed by hypernatremia, body mass loss, hyporexia, and increased activity of vasopressinergic and oxytocinergic neurons compared to controls. These results were associated with liver mass loss, decreased glycaemia, and increased cholesterolemia. Additionally, increased VO2 and a decreased respiratory exchange ratio indicated reduced carbohydrate consumption and potentially increased protein use during dehydration. Thus, skeletal muscle protein metabolism was evaluated due to its high protein content. In the oxidative muscles of the WD group, total and proteasomal proteolysis increased, which was associated with decreased Akt-mediated intracellular signaling. Interestingly, there was an increase in fiber cross-sectional area, likely due to higher muscle water content caused by increased intracellular osmolality induced by protein catabolism products. Conversely, no changes were observed in protein turnover or water content in glycolytic muscles. These findings suggest that short-term WD imposes a pro-catabolic state, depleting protein content in skeletal muscle. However, skeletal muscle may respond differently to dehydration based on its phenotype and might adapt for a limited time.

Exploitation of Biobased Co-Product Streams; Contributing to European Protein Demands Using Ethanol Plant Derived Co-Products

Abstract Europe is short of high-quality protein for animal feed, being only 29% self-sufficient in high-quality protein and relies heavily on imported proteins. But consumer demand is shifting towards more sustainable, home-grown sources of plant-based protein. This presents a significant challenge for the animal feed industry. Establishing a new plant protein crop for Europe would take a decade to create elite, geographically adapted varieties, adopt new agronomic practices, and establish appropriate supply chains. Approximately 9 million tons of European non-GM grains (maize, wheat, barley and rye) are converted annually by fermentation into bioethanol, a renewable fuel. The residue of the grain is currently used as medium-quality protein animal feed (distillers dried grains and solubles (DDGS; approx. 30% crude protein). Mechanically processing to separate the protein from the fibre fraction produces a Super-Pro (>50%crude protein concentration). The process produces commercially viable quantities of high-quality, non-GM grain protein, that can be used as an alternative to imported soybean products, in a time frame significantly ahead of establishing alternative agronomies or developing alternative novel proteins. Processing technology can transform low-quality co-product steams into high-quality alternative animal feed products and merits further investigation as a means of producing sustainable animal feed. Re-engineering grain bioethanol plants into biorefineries allows them to produce a super high protein (>50% crude protein) feed material concurrently with bioethanol, a renewable fuel. Information © The Authors 2024

Relationships Between Photosynthetic Efficiency and Grain Antioxidant Content of Barley Genotypes Under Increasing Nitrogen Rates

Nitrogen fertilization may affect the functioning of photosynthesis as well as the chemical composition and antioxidant potential of cereal grains. Little is known about the relationship between the efficiency of photosynthesis and the content of phenolic compounds in barley grain, especially in conditions of varying nitrogen availability. In this regard, a field experiment was conducted to examine the responses of two primary barley genotypes with elevated phenolic compound content (TPC) in grain and an intensive modern cultivar H. v. vulgare with high protein content to increasing nitrogen fertilization (rates of 0, 30, 60 and 90 kg N ha−1) during the study years, which differed in terms of hydrothermal conditions. The leaf greenness index (SPAD) and chlorophyll fluorescence parameters were evaluated on three occasions throughout the growing season. Following the harvest, the chemical composition of the grains, including phenolic acids, flavonoids and antioxidant potential, was evaluated. The antioxidant potential and chemical composition of the grain, including TPC and protein content, depended to the greatest extent on genetic and environmental factors, and only then on nitrogen fertilization. Nitrogen increased the TPC content and antioxidant capacity ABTS+ of the grains of all studied genotypes and the protein content in H. v. vulgare grain. Rates of 60 and 90 kg N ha−1 resulted in a significant increase in the SPAD, PIabs and Fv/Fm in BBCH 34 and 57. A positive correlation was confirmed between the SPAD and PIabs and the content of TPC and ABTS+ in the grain. The dependence of qualitative characteristics on the Fv/Fm was also demonstrated. The primary genotypes are characterized by a greater genetic potential for the synthesis of phenolic compounds than the modern cultivar H. v. vulgare. The synthesis of phenolic compounds, and thus their accumulation in the grain, is clearly stimulated by unfavorable environmental factors and moderate nitrogen rates and depends on the chlorophyll content in the leaves and the efficiency of photosynthesis. N fertilization has a beneficial effect on the content of phenolic compounds in grain resulting from the improvement in the SPAD and PIabs. The chemical composition of grain and the increase in antioxidant potential are determined by the Fv/Fm, which is low under hydrothermal stress conditions.

Low levels of apolipoprotein M are associated with increased risk of ventricular arrhythmias

Abstract Background Ventricular arrhythmia (VA) is a frequent cause of sudden cardiac death (SCD). Treatment to prevent SCD includes device therapy with implantable cardioverter defibrillator (ICD). Although the electrophysiology underlying VA have been described, the molecular understanding of VA risk remains unclear. Purpose To identify proteins that are associated with higher and lower risk of VA. Methods SMASH 1 Study was a prospective observational study of 490 patients treated with ICD. The plasma proteome was analyzed with the Olink Explore 384 Cardiometabolic platform at study inclusion, and patients were followed for mean 3.1± 0.7 years. VA events were defined as adjudicated ventricular fibrillation or sustained ventricular tachycardia registered from ICD recordings and clinical events were recorded from electronic health records. Protein concentrations were quantified relatively as normalized protein expression on a log2-transformed concentrations where a large number represents higher protein level in the sample. Results The mean age was 66±12 years, 83% were male and 51% had a primary prevention ICD-indication. Episode(s) of VA occurred in 137 patients (28%) during follow-up. In total, 353 distinct proteins were successfully analyzed in all patients. Among these, high B-type natriuretic peptide (BNP) and N-terminal pro-BNP (NT-proBNP) levels were associated with a higher risk of VA, while Apolipoprotein M (APOM) was the only protein significantly associated with a lower risk of VA after multiple testing using Benjamini-Hochberg correction (Figure, Panel A). After adjusting for age, sex, body mass index, coronary artery disease, heart failure, renal function, left ventricular ejection fraction and antiarrhythmic medication, only low APOM levels remained significantly associated with VA: HR 0.51 (95%CI 0.32-0.79] per log unit increase, p=0.003. Patients in the lowest quartile of APOM had a particularly high risk of incident VA: HR 2.26 (95%CI 1.60-3.19), p=3.61x10-6, compared to quartile 2-4 (Figure, Panel B). High APOM levels were also associated with a lower risk of HF hospitalizations (HR 0.25 [95%CI 0.16-0.40], p<0.001) and all-cause mortality (HR 0.36 [95%CI 0.22-0.60], p<0.001), and these associations remained significant in adjusted models. Conclusions Low levels of APOM are associated with a greater risk of incident VA, independently of established risk factors. Low APOM also predicted risk of HF hospitalizations and mortality in our cohort, which suggest protective cardiovascular effects of APOM. Figure: Association of individual proteomic measures with risk of ventricular arrhythmias (VA).A:Volcano plots of the associations of individual plasma proteins with incident VA. The dotted line represents the significance level at false discovery rate <0.05 and the red dots are significant proteins after multiple testing using Benjamini-Hochberg correction.B: Risk of VA by quartile of Apolipoprotein M.

The Protein/Carbohydrate Ratio in the Diet Affects Zootechnical Performance and the Regulation of Intermediary Metabolism in Juveniles Tambaqui (Colossoma macropomum).

The use of carbohydrates in animal feed is a way to save protein in the diet. This study evaluated the effect of protein/starch ratio on the performance, hepatic metabolism, and body composition of juvenile tambaqui (Colossoma macropomum). Six isoenergetic experimental diets were formulated containing three levels of digestible protein (P: 230, 260 and 290 g kg- 1) and two levels of starch (S: 180 and 280 g kg- 1): P23S18, P23S28, P26S18, P26S28, P29S18 and P29S28. Juveniles tambaqui (N = 288; initial weight = 30.0 ± 3.8 g) were fed experimental diets for 90 days and the zootechnical performance was evaluated. Nine fish from each treatment group were sampled to determine somatic indices, blood metabolites, tissue energy reserves, body composition and activity of key hepatic enzymes. Blood triglycerides, hepatosomatic index, liver and muscle lipids, and hepatic glycogen increased significantly with starch while blood glucose, triglycerides and cholesterol, muscle lipids were significantly impacted by protein levels. Fish-fed S28 had increased feed intake (1714-1829 g; p < 0.0001) and fish-fed diet P26S18 had a higher protein efficiency rate (23.62%; p = 0.0356). Fish fed P23 had lower crude protein retention in the carcass (p = 0.0018) and high lipogenesis and lipid deposition in the muscle (p = 0.0069). These results suggest that dietary carbohydrates have a protein-sparing effect. Tambaqui adapted well to diets containing starch, even at higher levels, resulting in an overall increase in lipogenesis from S18 to S28. Thus, we recommend P26S18 as a cost-effective feed that ensures both zootechnical performance and quality of the final product.

Secretomes from human epicardial adipocytes modulate atrial lymphatics and atrial fibrosis

Abstract Background Cardiac lymphatics maintains myocardial fluid and immune cell homeostasis. Downregulation of cardiac lymphatics leads to cardiac dysfunction through impairment of inflammation and edema. On the other hand, it is well known that adipose tissue accumulation causes lymphatic dysfunction and impairs lymphedema. Purpose We evaluated the expression of atrial lymphatics in human left atrial appendage (LAA) histologically and biologically. Subsequently, we examined secretomes from epicardial adipose tissue (EAT)-derived adipocytes and validated if they change atrial lymphatics expression using ex vivo Organo-culture system. Methods Human LAA samples were collected from forty-two patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=16, 73.1±10.4 years), the paroxysmal AF (PAF) group (n=13, 69.8±12.6 years) and the persistent AF (PeAF) group (n=13, 71.2±6.5 years). Human EAT were also excised from patients with and without AF (SR; n=5, AF; n=11) during cardiovascular surgery. Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation and maturation to adipocytes, cell culture supernatant (CCS) was collected, and was dropped onto the epicardial side of left atrial tissues excised from 8 weeks old male rats for 7 days using an ex vivo organo-culture system. Results In human LAA samples, mRNA expression of lymphangiogenesis-related genes (LYVE1, PROX1, VEGFC, and VEGFR3) was significantly lower in the PeAF group compared to the SR group (p=0.0370, &amp;lt;0.0001, 0.0157, and 0.0009). The number of LYVE1-positive atrial lymphatic endothelial cells (LECs) was also significantly lower in the PeAF group compared to the SR group and the PAF group (p=0.0186 and 0.038). There was a negative correlation between the number of LYVE1-positive atrial LECs and the area percentage of atrial myocardial fibrosis (r=-0.5350, p&amp;lt;0.001). In the experiment using organo-culture system, lymphangiogenesis-related genes expression (Lyve1, Prox1, Vegfc, and Vegfr3) of rat atria treated with CCS of adipocytes from AF patients was significantly lower than those from SR patients (p=0.0391, 0.0100, 0.0034, and 0.0338). Furthermore, the CCS of adipocytes from AF patients contained higher protein levels of leptin, which is known to suppress lymphangiogenesis (p=0.0357). Leptin treatment (100ng/ml) on rat atria also decreased lymphangiogenesis-related genes expression such as Lyve1, Prox1, Vegfc, and Vegfr3 (p=0.0413, &amp;lt; 0.0001, &amp;lt;0.0001, and 0.0190). Conclusions Our study with human LAA demonstrated that atrial lymphatics interact with atrial myocardial fibrosis/progression of AF. Furthermore, our findings indicate that secretomes from EAT-derived adipocytes regulate atrial lymphatics expression, and leptin plays an important role in modulating atrial lymphangiogenesis.

Takotsubo syndrome induced oxidative stress and endothelial dysfunction in left ventricle of rat model: association with NADPH oxidases/SGLT2 pro- oxidant pathway

Abstract Background Takotsubo syndrome (TTS) is characterized by apical ballooning predominantly affecting post-menopausal women following extreme physical and/or emotional stress. The phenotype of TTS is associated with akinetic apex and hyperkinetic base of the left ventricle (LV). Presently, there are no targeted therapies for TTS that can effectively reduce LV impairment. Empagliflozin (EMPA), an SGLT2 inhibitor, has shown considerable clinical benefits in managing heart failure. However, its specific effects and underlying mechanisms in treating TTS remain unclear. Purpose To explore the pathophysiology of TTS in the LV of rats, with a particular focus on the potential role of SGLT2, and to elucidate the underlying mechanisms involved. Methods Female Sprague Dawley rats (250-300 g) were allocated into four groups: a control group (n=8), a group treated with EMPA (30 mg/kg/day) (n=8), a group receiving ISO (Isoproterenol) at 100 mg/kg/day (n=18), and a group receiving both ISO and EMPA (IE) at 30 mg/kg/day (n=18). EMPA was administered orally through food two weeks prior to ISO injection. Echocardiography was conducted at 6 and 24 h, and on day 3. On day 3, rats were euthanized, and their organs were collected. Gene expression of markers was assessed using RT-qPCR, protein expression via immunofluorescence (IF) staining, and oxidative stress using dihydroethidium. Results In the ISO group, the success rate of induced Takotsubo syndrome (TTS) was 53% (10 out of 18 rats), with a mortality rate of 5%. Conversely, in the EMPA-treated group, TTS occurred in 22% of rats (4 out of 18) with no mortality observed. Significant reductions in the area of left ventricular (LV) ballooning and ejection fraction were noted in the IE group compared to the ISO group. Additionally, increased levels of reactive oxygen species (ROS) were observed in the LV of ISO rats, which were normalized by EMPA treatment in the IE rats. Furthermore, the ISO group exhibited higher protein expressions of CD68, SGLT2, and VCAM-1, which were attenuated by EMPA treatment. The mRNA expression of CYBA (p22phox) and NOX4, both representing subunits of NADPH oxidases, along with VCAM1, ICAM1, NOS2 (iNOS) and SELE (e-selectin) gene, endothelial activation marker, and TGF-β1, a fibrotic marker, showed elevated levels in the apex part of the left ventricle (LV) in the ISO group; these levels were significantly lower in the IE group. Conclusions These findings suggest that TTS is associated with oxidative stress-mediated endothelial dysfunction, inflammation, and pro-fibrotic responses. Moreover, SGLT2 inhibition appears to mitigate the pathology and progression of the syndrome. Therefore, SGLT2 inhibition emerges as an appealing and novel therapeutic approach for the treatment of TTS.

Dietary Administration Effects of Microbial Exopolysaccharide from Bacillus subtilis P1 on Growth Performance and Immunity in Nile Tilapia (Oreochromis niloticus)

Exopolysaccharide (EPS)-producing bacteria were isolated from the intestines of freshwater fish as prebiotics. Among the isolates, Bacillus sp. P1 was the potent EPS producer, with a high EPS production, and was then identified as Bacillus subtilis P1 based on 16S rRNA and biochemical characteristics. The produced microbial EPS was characterized by its functional groups by FTIR, showing a 90.20% correlation to inulin, while the EPS molecular weight was approximately 105 Da with a high PDI (&gt;1.5). Moreover, the EPS from B. subtilis P1 was assessed for prebiotic properties by growing probiotic bacteria, and significant cell growth occurred in sugar-free TSB with 0.5% EPS (p &lt; 0.05). EPS exhibited approximately 55.37% DPPH radical scavenging at 20 mg/mL and inhibited certain fish pathogens at 400 μg/mL (10–13 mm inhibition zone). Low EPS cytotoxicity was confirmed (&lt;1% hemolysis) prior to use as immunobiotics in Nile Tilapia (Oreochromis niloticus) diet supplementation. Nile tilapia growth and immune response were monitored after feeding with control (C), basal diet (BD), and treatment (T): BD + EPS 5 g/kg. In the T-group, the weight gain (WG), % specific growth rate (SGR), and average daily gain (ADG) significantly increased compared to the control (p &lt; 0.05) by week 4, with no negative effects on blood chemistry parameters. Lysozyme activity and respiratory burst activity in T-groups were significantly higher than the control (p &lt; 0.05), with a 50% RLP survival rate of Nile Tilapia infected by Aeromonas veronii in the T-group after a two-week challenge. On the other hand, the proximate analysis revealed higher protein content in the T-group. These findings suggest that EPS from B. subtilis P1 in fish diets efficiently supports fish growth and stimulates innate immune response in Nile Tilapia, thus showing potential as the immunobiotics for aquaculture.

Biochemical characterization of a recombinant laccase from Halalkalibacterium halodurans C-125 and its application in the biotransformation of organic compounds.

This study aimed to produce an engineered recombinant laccase from extremophilic Halalkalibacterium halodurans C-125 (Lac-HhC-125) with higher protein yield, into a more active conformation and with properties that meet the fundamental needs of biotechnological application. The rLac-HhC125 was partially purified by size exclusion chromatography and concentrated by ultrafiltration (10kDa) with a yield of 57.6%. Oxidation reactions showed that adding 2mM CuSO4 to the assay solution led to activating the laccase. To increase its initial activity, the rLac-HhC125 was treated at 50°C for 20min before the assays, improving its performance by fourfold using the syringaldazine as a substrate. When treated with EDTA, methanol, ethanol, and DMSO, the rLac-HhC125 maintained more than 80% of its original activity. Interestingly, the acetonitrile induced a twofold activity of the rLac-HhC125. The putative rLac-HhC125 demonstrated a capability of efficient transformation of different organic compounds at pH 6, known as dye precursors, into coloured molecules. The rLac-HhC125 was active at high temperatures and alkaline pH, exhibited tolerance to organic solvents, and efficiently transformed different hydroxy derivatives into coloured compounds, which indicates that it can be used in various biotechnological processes.

Comparison of Nutritional and Medicinal Ingredients Between Ganoderma leucocontextum and G. lucidum

The genus Ganoderma is a widely used medicinal fungus in East Asia. The main medicinal components are triterpenoids, polyphenols, and polysaccharides. Bitterness is an important commercial trait for Ganoderma. White Lingzhi (G. leucocontextum) is less bitter. But the characteristics of its nutritional and medicinal ingredients are still unclear, which undoubtedly limits its commercialization. In this study, the medicinal ingredients of Lingzhi and white Lingzhi were extracted and quantified. The structure and antioxidant activities of purified polysaccharides were determined. At the same time, their nutritional differences were compared. White Lingzhi contains more medicinal ingredients and its polysaccharide is more active. The higher protein content may be one of the reasons for weaker bitterness in white Lingzhi. The nutritional and medicinal traits of white Lingzhi were described for the first time in this study, which provides fundamental knowledge to support the development of white Lingzhi.

A multi-domain snail metallothionein increases cadmium resistance and fitness in Caenorhabditis elegans

Metallothioneins (MTs) are a family of mostly low-molecular weight, cysteine-rich proteins capable of specific metal-ion binding that are involved in metal detoxification and homeostasis, as well as in stress response. In contrast to most other animal species which possess two-domain (bidominial) MTs, some gastropod species have evolved Cd2+-selective multidomain MTs (md-MTs) consisting of several concatenated β3 domains and a single C-terminal β1 domain. Each domain contains three-metal ion clusters and binds three metal ions. The terrestrial snail Alinda biplicata possesses, among other MT isoforms, an md-MT with nine β3 domains and a C-terminal β1 domain (termed 10md-MT), capable of binding up to 30 Cd2+ ions per protein molecule. In the present study, the Alinda biplicata 10md-MT gene and a truncated version consisting of one β3 domain and a single C-terminal β1 domain (2d-MT) were introduced into a Caenorhabditis elegans knock-out strain lacking a native MT gene (mtl-1). The two snail MT constructs consistently increased Cd2+ resistance, and partially improved morphological, life history and physiological fitness traits in the nematode model host Caenorhabditis elegans. This highlights how the engineering of transgenic Caenorhabditis elegans strains expressing snail MTs provides an enhancement of the innate metal detoxification mechanism and in doing so provides a platform for enhanced mechanistic toxicology.

PREPARATION AND NUTRITIONAL EVALUATION OF ENRICHED JAM MADE FROM BLENDS OF APPLE, STRAWBERRY AND GRAPES

The aim of this study is to prepare an acceptable jam from blends of Apple, Strawberry and Grapes. Jams were produced from blends of Apple, Strawberry and Grapes in the following ratios 30:20:10:40 (A= 30g apple, 20g strawberry, 10g grapes and 40g of sugar) and 35:10:15:40 (B= 35g apple, 10g strawberry, 15g grapes and 40g of sugar) respectively. The pulp mixtures were combined with 40g of sugar. Mixed fruit jam and strawberry jam were used as commercial control labelled as sample C and D respectively. Standard methods were used for the determination of proximate composition, sugar content, phytochemical constituents and sensory evaluation of the Jam. The proximate composition indicated that sample A (15.31±0.12) and B (16.86±0.03) had a moisture content lower than the commercial control C (27.05±0.03) and D (27.60±0.45). The ash, fiber and lipid contents showed no significant difference in the jams produced and the commercial controls. Sample A and B had higher protein 29.58±0.02 and 27.41±0.01 as compared to the commercial controls C and D (24.62±0.02 and 25.15±0.01). No significant (P&gt;0.05) difference was observed in the glucose composition of the sample A and B and the commercial controls C and D. Fructose content in sample A (2.74±0.21) was higher than sample B (1.94±0.01) and the commercial controls C (1.85±0.00) and D (1.42±0.00). Qualitative testing for flavonoids, terpenoids, cardiac glycoside and tannin revealed potential good health benefits. The sensory evaluation revealed that sample A which comprises of 30% apple, 20% strawberry, 10% grapes and 40% sugar was rated best...

Histamine stimulates human microglia to alter cellular prion protein expression via the HRH2 histamine receptor

Although the cellular prion protein (PrPC) has been evolutionarily conserved, the role of this protein remains elusive. Recent evidence indicates that PrPC may be involved in neuroinflammation and the immune response in the brain, and its expression may be modified via various mechanisms. Histamine is a proinflammatory mediator and neurotransmitter that stimulates numerous cells via interactions with histamine receptors 1-4 (HRH1-4). Since microglia are the innate immune cells of the central nervous system, we hypothesized that histamine-induced stimulation regulates the expression of PrPC in human-derived microglia. The human microglial clone 3 (HMC3) cell line was treated with histamine, and intracellular calcium levels were measured via a calcium flux assay. Cytokine production was monitored by enzyme-linked immunosorbent assay (ELISA). Western blotting and quantitative reverse transcription-polymerase chain reaction were used to determine protein and gene expression of HRH1-4. Flow cytometry and western blotting were used to measure PrPC expression levels. Fluorescence microscopy was used to examine Iba-1 and PrPC localization. HMC3 cells stimulated by histamine exhibited increased intracellular calcium levels and increased release of IL-6 and IL-8, while also modifying PrPC localization. HMC3 stimulated with histamine for 6 and 24 hours exhibited increased surface PrPC expression. Specifically, we found that stimulation of the HRH2 receptor was responsible for changes in surface PrPC. Histamine-induced increases in surface PrPC were attenuated following inhibition of the HRH2 receptor via the HRH2 antagonist ranitidine. These changes were unique to HRH2 activation, as stimulation of HRH1, HRH3, or HRH4 did not alter surface PrPC. Prolonged stimulation of HMC3 decreased PrPC expression following 48 and 72 hours of histamine stimulation. HMC3 cells can be stimulated by histamine to undergo intracellular calcium influx. Surface expression levels of PrPC on HMC3 cells are altered by histamine exposure, primarily mediated by HRH2. While histamine exposure also increases release of IL-6 and IL-8 in these cells, this cytokine release is not fully dependent on PrPC levels, as IL-6 release is only partially reduced and IL-8 release is unchanged under the conditions of HRH2 blockade that prevent PrPC changes. Overall, this suggests that PrPC may play a role in modulating microglial responses.

MDA5 Is a Major Determinant of Developing Symptoms in Critically Ill COVID-19 Patients.

Apart from the skin and mucosal immune barrier, the first line of defense of the human immune system includes MDA5 (ifih1 gene) which acts as a cellular sensor protein for certain viruses including SARS-CoV-2. Upon binding with viral RNA, MDA5 activates cell-intrinsic innate immunity, humoral responses, and MAVS (mitochondrial antiviral signaling). MAVS signaling induces type I and III interferon (IFN) expressions that further induce ISGs (interferon stimulatory genes) expressions to initiate human cell-mediated immune responses and attenuate viral replication. SARS-CoV-2 counteracts by producing NSP1, NSP2, NSP3, NSP5, NSP7, NSP12, ORF3A, ORF9, N, and M protein and directs anti-MDA5 antibody production presumably to antagonize IFN signaling. Furthermore, COVID-19 resembles several diseases that carry anti-MDA5 antibodies and the current COVID-19 vaccines induced anti-MDA5 phenotypes in healthy individuals. GWAS (genome-wide association studies) identified several polymorphisms (SNPs) in the ifih1-ifn pathway genes including rs1990760 in ifih1 that are strongly associated with COVID-19, and the associated risk allele is correlated with reduced IFN production. The genetic association of SNPs in ifih1 and ifih1-ifn pathway genes reinforces the molecular findings of the critical roles of MDA5 in sensing SARS-CoV-2 and subsequently the IFN responses to inhibit viral replication and host immune evasion. Thus, MDA5 or its pathway genes could be targeted for therapeutic development of COVID-19.

The Dsc ubiquitin ligase complex identifies transmembrane degrons to degrade orphaned proteins at the Golgi

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition.

Effects of soybean insoluble dietary fiber and CaCl2 on the structure and properties of low-moisture extruded products.

Texturized vegetable protein is currently a leading alternative to animal meat. This study examined the effects of soybean insoluble dietary fiber (SIDF) (0% to 20%) and CaCl2 (0% to 1%) on the structure and properties of extruded products made from a soybean protein isolate-wheat gluten (SPI-WG) composite. The study showed that SIDF (4% to 8%) increased the viscosity of extruded products, enhanced their specific mechanical energy, and improved their rehydration rate and tensile strength compared with a control group. The rehydration rate of the extruded products reached a maximum value of 331.67% in the 8% SIDF, 0.5% CaCl2 groups. The addition of excess SIDF prevented the cross-linking of protein molecules to form a loose network structure. Analysis of the infrared spectrum and intermolecular forces showed that physical interactions between fibers and proteins were the dominant forces, with hydrophobic interactions and hydrogen bonds primarily maintaining the structure of the extruded products. The addition of CaCl2 (0.5%) led to protein aggregation and further improved the rehydration and tensile strength of extruded products. Soybean insoluble dietary fiber can improve the rehydration rate and quality of extruded products. The addition of CaCl2 mitigated the weakening of the protein structure caused by excess SIDF. These results provide a basis for the improvement of the quality of low-moisture-extruded texturized vegetable protein products with a high dietary fiber concentration and a high rehydration rate. © 2024 Society of Chemical Industry.

Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L.

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters—such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride—were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa.

Impacts of Protein and Energy Levels on Rumen Fermentation and Microbial Activity Under Different Incubation Temperatures

This study aimed to explore the effects of different incubation temperatures on ruminal fermentation and rumen microorganisms and determine the appropriate protein and energy levels to enhance microbial protein synthesis using an in vitro system. Rumen inoculum was collected from two fistulated Holstein heifers (trial 1: BW: 652.3 kg ± 25.2; trial 2: BW: 683.3 kg ± 30.2) and assessed using a closed-batch culture system. The experimental model employed a 2 × 5 factorial arrangement using incubation temperatures set to 39 and 41 °C, with protein levels set to 12.0, 13.5, 15.0, 16.5, and 18.0% of DM in trial 1 or with energy levels set to 2.4, 2.5, 2.6, 2.7, and 2.8 Mcal/kg of DM in trial 2. The data were analyzed using the MIXED procedure. The results showed increased (p &lt; 0.05) NH3-N concentrations and total volatile fatty acids (TVFAs) with higher incubation temperatures, while the liquid-associated bacterial (LAB) amounts decreased (p &lt; 0.05) in trials 1 and 2. The interaction between the energy level and incubation temperature affected (p &lt; 0.05) the LAB protein levels in trial 2. Higher protein levels led to increased (p &lt; 0.05) NH3-N and acetate concentrations, but it decreased (p &lt; 0.05) the propionate percentage. Conversely, higher energy levels decreased (p &lt; 0.05) the amount of acetate and increased the propionate concentration, altering the acetate-to-propionate ratio. However, no interaction involving TVFA and LAB was observed between the incubation temperature and the protein or energy levels. Changes in the NH3-N, TVFAs, and LAB protein amounts were observed under different incubation temperatures and energy levels. In conclusion, these findings provide insight into the metabolic adaptation under different ruminal temperatures and the impacts of dietary adjustments on rumen fermentation and microbial activity. However, there are limitations to replicating the complex physiological responses that occur within the whole body solely through in vitro experiments.

Lefamulin dosing optimization using population pharmacokinetic and pharmacokinetic/pharmacodynamic assessment in Chinese patients with community-acquired bacterial pneumonia

PurposeLefamulin is the first pleuromutilin antibiotic approved for the treatment of community-acquired bacterial pneumonia (CABP). However, the pharmacokinetic/pharmacodynamic (PK/PD) characteristics in Chinese CABP patients are not fully understood. This study aimed to evaluate its microbiological efficacy against Streptococcus pneumoniae and Staphylococcus aureus via PK/PD analysis.MethodsThe population PK (PopPK) model, established with foreign data was validated using data from Chinese CABP patients. PK/PD analysis was conducted for the intravenous administration of 150 mg q12 h for 1-h, 1.5-h and extended 2-h infusion. Oral administrations of 600 mg q12 h were assessed, considering original and higher plasma protein binding.ResultsLefamulin displayed similar PK characteristics in both Chinese and Western populations. The PopPK model effectively predicted lefamulin concentrations in Chinese CABP patients. Under the dosage regimen of 150 mg q12 h via intravenous infusion for 1/1.5/2 h, the probability of target attainments reached 98% at the minimum inhibitory concentration for both 90% S. pneumoniae and S. aureus, considering both original and higher protein binding rates. It is advisable to extend the infusion duration from 1/1.5 h–2 h to minimize the risk of adverse effects at the infusion site. Regardless of fasted or fed conditions, the PTAs for 600 mg q12 h lefamulin via oral administration proved comparable to those for intravenous administration.ConclusionThis study proved that intravenous and oral administrations of lefamulin can reach preclinical PK/PD targets of S. pneumoniae and S. aureus. These findings support the optimal use of lefamulin for the safe and effective treatment of Chinese CABP patients.

Cellular Characteristics and Protein Signatures of Human Adipose Tissues from Donors With or Without Advanced Coronary Artery Disease

Background/Objectives: Perivascular adipose tissue (PVAT) exerts a paracrine effect on blood vessels and our objective was to understand PVAT molecular signatures related to cardiovascular disease. Methods: We studied two groups: those undergoing mitral valve repair/replacement (VR, n = 16) and coronary artery bypass graft (CABG, n = 38). VR donors did not have coronary artery disease, whereas CABG donors had advanced coronary artery disease. Clinical and tissue pathologies and proteomics from adipose tissue were assessed. Results: Donors undergoing VR had a lower body mass index (p = 0.01), HbA1C (p = 0.0023), and incidence of diabetes (p = 0.022) compared to CABG. VR donors were overall healthier, with higher cardiac function compared to CABG donors, based on ejection fraction. Although adipose histopathology between groups was not markedly different, PVAT had smaller and more adipocytes compared to subcutaneous adipose tissues. These differences were validated by whole specimen automated morphological analysis, and anisotropy analysis showed small (2.8–7.5 μm) and large (22.8–64.4 μm) scale differences between perivascular and subcutaneous adipose tissue from CABG donors, and small scale changes (2.8–7.5 μm) between perivascular and subcutaneous adipose tissue from VR donors. Distinct protein signatures in PVAT and subcutaneous adipose tissue include those involved in secretion, exosomes and vesicles, insulin resistance, and adipocyte identity. Comparing PVAT and subcutaneous adipose tissue from CABG donors, there were 82 significantly different proteins identified with log fold change ≥ 0.3 or ≤−0.3 (p &lt; 0.05). Using this threshold, there were 36 differences when comparing PVAT and subcutaneous adipose tissue from VR donors, 58 differences when comparing PVAT from CABG or VR donors, and 55 when comparing subcutaneous adipose tissue from CABG vs. VR donors. Conclusions: Routine histopathology cannot differentiate between PVAT from donors with or without coronary artery disease, but multiscale anisotropy analysis discriminated between these populations. Our mass spectrometry analysis identified a cohort of proteins that distinguish between adipose depots, and are also associated with the presence or absence of coronary artery disease.